DE60110094T2 - DEVICE AND METHOD FOR THE AUTOMATED SYNTHESIS OF OLIGOSACCHARIDES - Google Patents

Abstract

One aspect of the present invention relates to an apparatus for the efficient synthesis of oligosaccharides on a solid support, e.g., formed by subunit addition to terminal subunits immobilized on solid-phase particles. In certain embodiments, the apparatus of the present invention is used in combinatorial methods, e.g., as described herein, of synthesizing oligosaccharides.

Description

The The present invention relates to an automatic apparatus and method Synthesis of oligosaccharides.

State of the art

biopolymers like polypeptides and polynucleotides are routinely using Solid phase method synthesized in which polymer subunits progressively to a growing polymer chain immobilized on a solid support is added become. For Polynucleotides and polypeptides may be general synthetic Procedure with commercially available Synthesizers performed be the biopolymers with defined sequences in an automated or semi-automated fashion. Commercially available Synthetizer enable but not the efficient synthesis of oligosaccharides; typically are the yields of oligosaccharides using commercially available Devices are synthesized, bad.

The Glycosylation reaction is one of the most thoroughly studied transformations in organic chemistry. In the most general sense is glycosylation the formation of an acetate which combines two sugar units. The majority of the glycosylating agents follow a similar one Reaction mechanism. The anomeric substituent acts as a Leaving group, creating an electrophilic intermediate becomes. The reaction of these species with a nucleophile, typically a hydroxyl group to the formation of a glycosidic bond. The reaction can be over a Variety of intermediates, depending on the nature of the Leaving group, the activating reagent and the solvent used occur.

Glycosyl trichloroacetimidates, thioglycosides, glycosyl sulfoxides, glycosyl halides, glycosyl phosphites, n-pentenyl glycosides and 1,2-anhydro sugar are among the most reliable glycosyl donors (see 1 ). Despite the abundance of available glycosylating agents, not a single process has distinguished itself as a universal donor. In contrast to peptide and oligonucleotide synthesis, the inherent differences in monosaccharide structures make it unlikely that a common donor will interspersed. Instead, individual donors will find use in the construction of certain glycosidic linkage classes.

Chemical solid phase synthesis

Solution phase oligosaccharide synthesis remains a slow process because of the need for the iterative coupling and deprotection steps with purification at each step along the way. To alleviate the need for repetitive cleaning operations, solid phase techniques have been developed. Two methods are available for solid phase oligosaccharide synthesis (see 2 and 3 ). The donor-bonded process combines the first sugar with the polymer through the non-reducing end of the monomer unit ( 2 ). The polymer-bound sugar is then converted to a glycosyl donor and treated with an excess of acceptor and activator. The productive coupling leads to polymer-bound disaccharide formation while the degradation products remain bound to the resin. Extension of the oligosaccharide chain is achieved by converting the newly added sugar moiety to a glycosyl donor and repeating the above cycle. Since most donor species are highly reactive, using the donor-bonded process increases the possibility of forming polymer-bound by-products.

Alternatively, acceptor-linked strategies have found considerable use in solid-phase oligosaccharide synthesis ( 3 ) found. In this approach, the first sugar is attached to the polymer at the reducing end. The removal of a unique protecting group on the sugar gives a polymer-bound acceptor. The reactive glycosylating agent is delivered in solution and the productive coupling results in polymer-linked oligosaccharides while washing away the undesirable by-product caused by donor degradation. Removal of a unique protecting group on the polymer-linked oligosaccharide releases another hydroxyl group for extension.

While the benefits of the donor-linked method of Danishefsky and co-workers have been demonstrated, the most popular and generally applicable method for the synthesis of oligosaccharides on a polymer support remains the acceptor-linked strategy. For an overview, see PH Seeberger, SJ Da nishefsky, Acc. Chem. Res., 31 (1998), 685. The ability to use an excess of glycosylating agent in solution to drive the reaction towards completion has led to widespread use of this method. All of the above glycosylating agents have been used with the acceptor-linked method with varying degrees of success.

It There is an increasing demand for automated synthesis of oligosaccharides. It is, for example, often under investigation the structure-function relationships, which involve sugar of interest, a mixture of Oligosaccharides that have different residues on a given Position or in the anomeric configuration differentiate a glycosidic bond. As further Example can Oligosaccharides which are a desired Activity, like a high binding affinity to a given receptor or antibody by (a) generating a big one Number of random sequence oligosaccharides, and (b) screening of these oligosaccharides to one or more oligosaccharides with the desired binding affinity be identified.

A current device for synthesizing oligosaccharides both in terms of number and quantity of oligosaccharides, which can be synthesized limited. These restrictions have the availability of oligosaccharides for limited to structural analysis as well as to competitions.

Summary of the invention

In In a first aspect, the present invention provides a device for the automated solid-phase synthesis of oligosaccharides which Comprising: a reaction vessel having at least one insoluble one Resin beads, at least one donor vessel with a Sacchariddonatorlösung, at least one activator vessel with a Assets Torre Agen solution at least one deblocking vessel with a deblocking, at least one solvent vessel with a Solvent; a solution transfer system, which is capable of the saccharide donor solution, activator reagent solution, deblocking reagent solution and the solvent transferred to; and a computer for controlling the solution transfer system. The / The insoluble resin beads can consist of an octenediol-functionalized resin.

A Device according to the invention can for used the efficient synthesis of oligosaccharides on a solid support be, the z. B. by the addition of subunits to terminal Subunits immobilized on solid phase particles are formed is. In certain embodiments For example, the device of the present invention will be combinatorial Method used, for example, as described herein for synthesizing of oligosaccharides.

The insoluble Resin beads, contained within the reaction vessel are, can a glycosyl acceptor over have attached an organic linker to the resin bead. Of the Organic linker may have glycosyl phosphate.

The Device may be a temperature control unit for regulating have the temperature of the reaction vessel. The temperature control unit can by the same computer, the Solution control system can be controlled, controlled and the internal temperature of the Measure reaction vessel. The Temperature control unit may be capable of the reaction temperature at a temperature between -80 ° C and + 60 ° C, preferably between -25 ° C and + 40 ° C. In one embodiment According to the invention, the reaction vessel can have a double-walled structure be which two cavities forms, wherein in the first cavity, the synthesis of the oligosaccharides takes place, and wherein the second cavity is a coolant of the temperature control unit contains. The double-walled structure of the reaction vessel may consist of glass.

The Saccharide donor solution in the donor vessel can a Monosaccharide donor solution be. The solution transfer system may be able to solve the problem while it is kept under an inert gas pressure to transfer.

Any / Donor vessel (s) can / a Solution, which comprises a glycosyl trichloroacetimidate and / or a glycosyl phosphate solution.

Any / Aktivatorgefäß (s) can / can a solution containing a Lewis acid, such as a silyl trifluoromethanesulfonate, such as a trimethylsilyl trifluoromethanesulfonate having.

Any / Deblocking vessel (s) can / can solution which may be sodium methoxide (which may be in methanol) or Having hydrazine.

Any / Solvent vessel (s) may be dichloromethane, Methanol and / or tetrahydrofuran included. A first solvent vessel may be dichloromethane contain a second solvent vessel, methanol and a third solvent vessel may be tetrahydrofuran contain.

The Device may further comprise at least one blocking vessel, which contains a blocking reagent solution. The blocking vessel (s) may have a solution contain, for example, a solution which contains benzyl trichloroacetimidate or a carboxylic acid. The carboxylic acid can be levulinic acid. In one embodiment contains a first solvent vessel dichloromethane, a second solvent vessel contains methanol and a third solvent vessel containing tetrahydrofuran; a fourth solvent vessel contains a Solution, which pyridine and acetic acid and a fifth Solvent vessel contains a 0.2 M solution of acetic acid in tetrahydrofuran. In another embodiment, a first donor vessel contains a solution which Glycosyl trichloroacetimidate, a second donor vessel containing a solution which a first glycosyl phosphate, a third donor vessel contains one Solution, which has a second glycosyl phosphate, at least one Activator vessel contains one Solution, which comprises trimethylsilyl trifluoromethanesulfonate, a first Deblocking vessel contains a Solution, which has hydrazine, a second deblocking vessel contains a Solution, which comprises sodium methoxide, a first solvent vessel contains dichloromethane, a second solvent vessel contains methanol and a third solvent vessel containing tetrahydrofuran, a fourth solvent vessel contains a Solution, which pyridine and acetic acid and a fifth Solvent vessel contains a 0.2 M solution of acetic acid in tetrahydrofuran.

In In a second aspect, the present invention provides a method for forming a carbon-heteroatom bond between a glycosyl donor and a substrate, which method in solution in Reaction vessel of a Device of the invention, a glycosyl donor, which a having reactive anomeric carbon, a substrate which has a heteroatom which carries a hydrogen, and an activating reagent combined, wherein the activating reagent activates the reactive anomeric carbon of the glycosyl donor, thereby forming a product having a carbon-heteroatom bond between the anomeric carbon of the glycosyl donor and the heteroatom of the substrate. The glycosyl donor, which is a reactive having anomeric carbon may be selected from the group consisting of Glycosyl phosphates, glycosyl phosphites, glycosyl trichloroacetimidates, Glycosyl halides, glycosyl sulfides, glycosyl sulfoxides, n-pentenyl glycosides and 1,2-anhydroglycosides be. The heteroatom, which carries a hydrogen of the substrate, can from the group consisting of oxygen, nitrogen and sulfur selected be.

In certain embodiments of the process of the present invention is the activating reagent a Lewis acid. In certain embodiments of the method of the present invention is the activating reagent Silyltrifluoromethansulfonat. In certain embodiments of the method In the present invention, the activating reagent is trimethylsilyl trifluoromethanesulfonate.

In certain embodiments of the process of the present invention, the substrate having a hydrogen bearing heteroatom is attached to a solid support via a covalent linker. In certain embodiments of the process of the present invention, the covalent linker is -O- (CH ₂ ) ₃ CH = CH (CH ₂ ) ₃ -O-. In certain embodiments of the process of the present invention, the solid support is a resin bead.

In certain embodiments of the process of the present invention, said reactive anomeric carbon glycosyl donor is attached to a solid support via a covalent linker. In certain embodiments of the process of the present invention, the covalent linker is -O- (CH ₂ ) ₃ CH = CH (CH ₂ ) ₃ -O-. In certain embodiments of the process of the present invention, the solid support is a resin bead.

In certain embodiments the method of the present invention is said substrate, which has a hydrogen-bearing heteroatom the group consisting of monosaccharides, oligosaccharides, polysaccharides and glycoconjugates selected.

In certain embodiments, a method of the present invention further comprises the steps of applying positive pressure or a vacuum to the reaction vessel of the device, whereby the flüs Sige phase is removed from the reaction vessel of the device, and the addition of solvent to the reaction vessel of the device.

In certain embodiments For example, a method of the present invention further includes the step treating the product in the reaction vessel of the device with a solution which has a reagent for removing the protection, whereby a protecting group is removed from the product to form a second product which is a hydrogen-bearing heteroatom wherein the second product is via a covalent linker to a solid support is bound.

In certain embodiments has a method of the present invention further in solution in the Reaction vessel of the device the step, a glycosyl donor, which is a reactive anomeric carbon, wherein the second product is a Having hydrogen bearing heteroatom with an activating reagent wherein the activating reagent is the reactive anomer Activated carbon of the glycosyl donor, creating a third Product is formed, which is a carbon-heteroatom bond between the anomeric carbon of the glycosyl donor and the heteroatom of the second product, wherein the third product via a Covalent linker is bound to a solid support.

In certain embodiments For example, a method of the present invention further comprises the step of Treating the product in the reaction vessel of the device with a solution which is a conversion reagent for producing a second Having a product which is a reactive anomeric carbon wherein the second product is via a covalent linker to a solid support is bound.

In certain embodiments has a method of the present invention further in solution in the Reaction vessel of the device the step of, a substrate which is a hydrogen-bearing heteroatom wherein the second product is a reactive anomeric carbon has to combine with an activating reagent, wherein the Aktivierungsreagens the reactive anomeric carbon of the second Product, thereby forming a third product, which is a carbon-heteroatom bond between the anomeric Carbon of the second product and the heteroatom of the substrate wherein the third product is via a covalent linker to a solid support is bound.

Brief description of the figures

1 represents certain commonly used glycosylating agents.

2 generally represents donor-bound solid-phase carbohydrate synthesis.

3 generally represents an acceptor-linked carbohydrate synthesis.

4 represents the major classes of repeating biooligomers responsible for the signal transduction process.

5 Figure 1 illustrates one embodiment of an automated oligosaccharide synthesizer according to the present invention.

6 Figure 4 illustrates another embodiment of an automated oligosaccharide synthesizer according to the present invention.

7 FIG. 10 illustrates one embodiment of a double-walled cooled reaction vessel according to the present invention. FIG.

8th Figure 2 illustrates a comparison of the 2-D NMR spectra of resin-bound and solution-phase pentasaccharides.

9 represents a fully protected phylo-alxin inducer (PE) glucan synthesized using the device of the present invention.

10 provides an overview of automated solid-phase synthesis of oligosaccharides.

11 provides an overview of the steps of an automated solid-phase synthesis of Oligosac charids using glycosyl phosphates.

12 Figure 1 illustrates a cycle of oligosaccharide synthesis on the solid support using glycosyl phosphates.

13 represents the automated synthesis cycle used in the synthesis of a hexasaccharide using glycosyl phosphates.

14 Fig. 12 illustrates the HPLC data for hexasaccharide synthesized using the apparatus of the present invention.

15 provides an overview of the steps in the automated synthesis of oligomannosides.

16 represents oligosaccharides synthesized using the methods and apparatus of the invention and the coupling cycle used for the synthesis of the oligosaccharides.

17 For example, the HR-MAS HMQC represents data for an oligosaccharide synthesized using both the device of the present invention and in solution.

18 Fig. 12 illustrates the HPLC data for heptamannoside synthesized using the apparatus of the present invention.

19 Figure 2 illustrates the automated synthesis of a decamannoside using trichloroacetimidate glycosyl donors.

20 represents the automated synthesis of Leishmania cap tetrasaccharides.

21 represents the automated synthesis of GlcA trisaccharides.

22 represents the automated synthesis of polyglucosamine.

Detailed description the invention

definitions:

For the expediency certain terms in the description, examples, and the connected claims determined here.

Of the Term "heteroatom" like him used herein, an atom of each element means that of carbon or hydrogen is different Preferred heteroatoms are boron, Nitrogen, oxygen, phosphorus, sulfur and selenium.

Of the Term "electron-withdrawing Group "is recognized in the art and means the tendency of a substituent Attracting valence electrons from neighboring atoms, d. h., in relation to the neighboring atoms, the substituent is electronegative. A quantification The level of electron-withdrawing ability is determined by the Hammett sigma () constant where. This known constant is in many references described, for example, J. March, Advanced Organic Chemistry, McGraw Hill Book Company, New York, (1977 Edition), pp. 251-259. The Values of Hammet's constant are generally for electron-donating groups ([P] = -0.66 for NH2) negative and for Electron withdrawing group ([P] = 0.78 for a nitro group) positive, where [P] indicates a para substitution. Examples of electron-withdrawing Groups count Nitro, Acyl, formyl, sulfonyl, trifluoromethyl, cyano, chloride, and the like. Examples of electron donating groups include amino, Methoxy and the like.

The term "alkyl" refers to a radical of a saturated aliphatic group, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cyclalkyl groups, and cyclalkyl-substituted alkyl groups. In preferred embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in the backbone (e.g., C ₁ -C ₃₀ for straight chain, C ₃ -C ₃₀ for branched chain), and more preferably 20 or less. Likewise, preferred cycloalkyls have 3-10 carbon atoms in their ring structure, and more preferably have 5, 6 or 7 carbons in their ring structure.

Of the Term "aralkyl" like him As used herein, an alkyl group refers to those having an aryl group (eg, an aromatic or heteroaromatic group) is.

Of the Term "alkenyl" and "alkynyl" unsaturated aliphatic groups, which in their length and possible substitution to the alkyls described above are analogous, but at least one double or triple bond included.

If the number of carbon atoms is not specified otherwise means a "low Alkyl "as well it is used herein, an alkyl group as defined above, but with one to ten carbons, more preferably from one to six carbon atoms in their main chain structure. Similarly, "lower ones Alkenyl "and" lower Alkynyl "similar Chain lengths. Preferred alkyl groups are lower alkyls. In preferred embodiments For example, a substituent referred to herein as alkyl is a lower alkyl.

The term "aryl" as used herein includes 5-, 6- and 7-membered single aromatic ring groups such as benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, Pyridazine and pyrimidine, and the like, containing from zero to four heteroatoms. Those aryl groups having heteroatoms in the ring structure may also be referred to as "aryl heterocycles" or "heteroaromatics". The aromatic ring may be substituted at one or more ring positions, with such substituents as described above, such as, for example, halo, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalky, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, Phosphonate, phosphinate, carbonyl, carboxyl, silyl, ethers, alkylthio, sulfonyl, sulfonamido, ketone, aldehydes, esters, heterocyclyl, aromatic and heteroaromatic radicals, -CF ₃ , -CN, or the like may be substituted. The term "aryl" also includes polycyclic ring systems having two or more cyclic rings in which two adjacent rings share two or more carbons (the rings are "fused rings") wherein at least one of the rings is aromatic, e.g. B, the other cyclic ring may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl and / or heterocyclyl.

The Terms ortho, meta, and para apply to 1,2-, 1,3-, and 1,4-disubstituted, respectively Benzene. For example, the names are 1,2-dimethylbenzene and ortho-dimethylbenzene to use synonymously.

The terms "heterocyclyl" or "heterocyclic group" refer to 3- to 10-membered ring structures, more preferably 3- to 7-membered rings whose ring structures contain one to four heteroatoms. Heterocycles can also be polycycles. Heterocyclyl groups include, for example, thiophene, thianthrene, furan, pyran, isobenzofuran, chromene, xanthene, phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine , Quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, chinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine, phenanthroline, phenazine, phenarsazine, phenothiazine, furazane, phenoxazine, pyrrolidine, oxolane, thiolane, oxazole, piperidine , Piperazine, morpholine, lactones, lactams, such as azetidinones, and pyrrolidinones, sultams, sultones, and the like. The heterocyclic ring may be substituted at one or more positions with such substituents as described above, such as halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate , Carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, an aromatic or heteroaromatic radical, -CF ₃ , -CN or the like.

The term "polycyclyl" or "polycyclic group" refers to two or more rings (ie, cyclic alkyls, cycloalkenyls, cycloalkynyls, aryls and / or heterocyclyls) in which two adjacent rings share two or more carbon atoms, e.g. For example, the rings are "condensed rings". Rings connected by non-adjacent atoms are called "bridged" rings. Each of the rings of the polycycles can be substituted by such substituents as described above, such as halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, Carboxyl, silyl, ester, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic radical, -CF ₃ , -CN, or the like.

As used herein, the term "nitro" means -NO ₂ ; the term "halogen" denotes -F, -Cl, -Br or -I; the term "sulfhydryl" means -SH; the term "hydroxyl" means -OH; and the term "sulfonyl" means -SO ₂ .

wobei R₉, R₁₀ und R'₁₀ jeweils unabhängig eine Gruppe, die durch die Valenzregeln zugelassen sind, darstellt.The terms "amine" and "amino" are recognized in the art and both refer to unsubstituted and substituted amines, e.g. For example, a radical that can be represented by the general formula:

wherein R ₉ , R ₁₀ and R _'10 each independently represent a group permitted by the valence rules.

wobei R₉ wie oben definiert ist, und R'₁₁ stellt ein Wasserstoff, ein Alkyl, ein Alkenyl oder -(CH₂)_m-R₈ dar, wobei m und R₈ wie oben definiert sind.The term "acylamino" is recognized in the art and refers to a radical that can be represented by the following general formula:

wherein R _{9 is} as defined above, and R _'11 represents a hydrogen, an alkyl, an alkenyl or - (CH ₂ ) _m -R ₈ , wherein m and R _{8 are} as defined above.

wobei R9, R10 wie oben definiert sind. Bevorzugte Ausführungsformen des Amids werden keine Imide beinhalten, welche instabil sein können.The term "amido" is recognized in the art as an amino-substituted carbonyl and includes a group which can be represented by the general formula

wherein R9, R10 are as defined above. Preferred embodiments of the amide will not include imides which may be unstable.

The term "alkylthio" refers to an alkyl group as defined above having a sulfur radical attached thereto. In preferred embodiments, the "alkylthio" radical is represented by one of -S-alkyl, -S-alkyl, -S-alkenyl, -S-alkynyl, and -S (CH ₂ ) _m -R ₈ wherein m and R _{8 are} as defined above.

wobei X eine Bindung ist oder Sauerstoff oder ein Schwefel darstellt, und R₁₁ stellt ein Wasserstoff, ein Alkyl, ein Alkenyl oder -(CH₂)_m-R₈ oder ein pharmazeutisch akzeptables Salz dar, R'₁₁ stellt ein Wasserstoff, ein Alkenyl oder -(CH₂)_m-R₈ dar, wobei m und R₈ wie oben definiert sind.The term "carbonyl" is recognized in the art and includes such radicals as represented by the general formula:

wherein X is a bond or oxygen or sulfur and R ₁₁ is hydrogen, alkyl, alkenyl or - (CH ₂ ) _m -R ₈ or pharmaceutically acceptable salt, R _'11 is hydrogen, alkenyl or - (CH ₂ ) _m -R ₈ , where m and R _{8 are} as defined above.

Where X is an oxygen and R ₁₁ or R _{'11 is} not hydrogen, the formula represents an "ester". Where X is an oxygen and R _{11 is} as defined above, the remainder herein refers to a carboxyl group, and especially when R _{11 is} hydrogen, the formula represents a "carboxylic acid". Where X is an oxygen and R _{'11 is} hydrogen, the formula represents a "formate". In general, where the oxygen atom of the above When X is sulfur and R ₁₁ or R _{'11 is} not hydrogen, the formula represents a "thiolester". Where X is sulfur and R _{11 is} hydrogen Where X is sulfur and R _{'11 is} hydrogen, the formula represents a "thiol formate". On the other hand, when X is a bond and R _{11 is} not hydrogen, the above formula sets one "Ketone" group. If X is a bond, and R _{11 is} hydrogen, the above formula represents an "aldehyde" group.

The terms "alkoxyl" or "alkoxy" as used herein refer to an alkyl group as defined above having an oxygen radical attached thereto. Representative alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy and the like. An "ether" consists of two hydrocarbons covalently linked to each other via an oxygen. Accordingly, the substituent of an alkyl which makes the alkyl an ether is or is an alkoxyl such as one of -O-alkyl, -O-alkenyl, -O-alkynyl, -O- (CH ₂ ) _m - R ₈ may be represented, where m and R _{8 are} as described above.

wobei R₄₁ ein Elektronenpaar, Wasserstoff, Alkyl, Cycloalkyl, oder Aryl ist.The term "sulfonate" is recognized in the art and includes a radical that can be represented by the general formula:

wherein R _{41 is} an electron pair, hydrogen, alkyl, cycloalkyl, or aryl.

The Terms triflyl, tosyl, mesyl, and nonaflyl are recognized in the art and refer to trifluoromethanesulfonyl, p-toluenesulfonyl, Methanesulfonyl, or Nonafluorbutansulfonyl groups. The terms Triflate, tosylate, mesylate, and nonaflate are recognized in the art and refer to trifluoromethanesulfonate ester, p-toluenesulfonate ester, Methanesulfonatester, and Nonafluorbutansulfonatester functional Groups or molecules, which contain such groups.

The Abbreviations Me, Et, Ph, Tf, Nf, Ts, Ms represent Methyl, ethyl, phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl, p-Toluensulfonyl or methanesulfonyl. A more extensive list of abbreviations, used by the person skilled in the art of organic chemistry will appear in the first issue of each volume of the journal of Organic Chemistry; This list is usually in a table entitled Standard List of Abbreviations. In the Abbreviations contained in this list, and all abbreviations used by the person skilled in the art of organic chemistry are hereby incorporated by reference.

wobei R₄₁ wie oben definiert ist.The term "sulfate" is recognized in the art and includes a radical that can be represented by the general formula:

wherein R _{41 is} as defined above.

The term "sulfonylamino" is recognized in the art and includes a moiety that can be represented by the following formula:

The term "sulfamoyl" is recognized in the art and includes a moiety that can be represented by the following formula:

wobei R₄₄ aus der Gruppe bestehend aus Wasserstoff, Alkyl, Alkenyl, Alkynyl, Cycloalkyl, Heterocyclyl, Aryl, oder Heteroaryl ausgewählt ist.The term "sulfonyl" is recognized in the art and includes a moiety that can be represented by the following formula:

wherein R _{44 is selected} from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl is selected.

wobei R₄₄ aus der Gruppe bestehend aus Wasserstoff, Alkyl, Alkenyl, Alkynyl, Cycloalkyl, Heterocyclyl, Aralkyl, oder Aryl ausgewählt ist.The term "sulfoxide" as used herein refers to a group which can be represented by the following formula:

wherein R _{44 is selected} from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aralkyl, or aryl.

A "selenoalkyl" refers to an alkyl group having a substituted selenium group attached thereto. Exemplary "selenium ethers" which may be substituted for an alkyl are selected from one of -Se-alkyl, -Se-alkenyl, -Se-alkynyl, and -Se- (CH ₂ ) _m -R ₇ wherein m and R _{7 are} as defined above.

analog Substitutions can on alkenyl and alkynyl groups are made, for example Aminoalkenyls, aminoalkynyls, amidoalkenyls, amidoalkynyls, iminoalkenyls, Iminoalkynyls, thioalkenyls, carbonyl-substituted alkenyls or Alkynyls produce.

As used herein, it is intended that the definition of each term, z. B. alkyl, m, n, etc, if he is more than once in any Structure occurs, dependent from its definition elsewhere in the same structure can be.

It is understandable, that substitution or substituted with the implicit provision implies that such a substitution in accordance with the approved valence of the substituted atom and the substituent takes place, and that the substitution becomes a stable compound, z. Which does not undergo spontaneous transformation such as rearrangement, cyclization or elimination experiences, leads.

As used herein, the term "substituted" is to be regarded as that he is all allowed Substituents of organic compounds. In the far Meaning include the permissible Substituents acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic Substituents of organic compounds. For exemplary substituents counting, for example, those described herein. The permissible substituents can one or more and the same or different for corresponding ones be organic compounds. For Purposes of this invention can the heteroatoms, such as nitrogen, hydrogen substituents and / or any permissible Substituents of organic compounds have described herein are those which satisfy the valences of the heteroatoms. This invention is not intended by the permissible Substituents of organic compounds in any way and Way limited to be.

The Phrase "protective group" as they are used herein means temporary substituents which are potentially reactive functional groups from unwanted chemical transformations protects. Examples of such protecting groups include esters of carboxylic acids, silyl ethers of alcohols, and acetals or ketals of aldehydes or ketones. The field of protecting group chemistry has been reviewed (Greene, T. W .; Wuts, P.G.M. Protective Groups in Organic Synthesis, 2nd Edition; Wiley; New York, 1991).

Certain Compounds of the present invention may be used in particular geometric or stereoisomeric forms exist. The present invention contemplates all such compounds, including cis and trans isomers, R and S enamers, Diastereomers, (D) -isomers, (L) -isomers, the racemic mixtures thereof, and other mixtures thereof, than within the scope of protection falling within the scope of the invention. additional asymmetric carbon atoms can in a substituent such as an alkyl group. All such isomers, as well as mixtures thereof, are intended in this Invention to be included.

For example, if a particular enantiomer of a compound of the present invention is desired, it can be prepared by asymmetric synthesis or by derivation with a chiral auxiliary additive where the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide the pure desired enantiomer. Alternatively, in cases where the molecule contains a basic functional group, such as amino or an acidic functional group, such as carboxyl, with a corresponding optically active acid or base, followed by fractionation of the diastereomers so formed Crystallization or by chromatographic co tel, which are known in the art, and then the recovery of the pure enantiomers.

Considered equivalents The compounds described above include compounds which otherwise correspond, and the same general characteristics have (for example acting as analgesics), one or Several simple variations of the substituents are made the effectiveness of the compound in binding to the sigma receptors not adversely affect. In general, the compounds of the present Invention are prepared by the methods described in the general Reaction schemes are shown, for example, described below are, or by modification thereof, by using easy available Starting materials, reagents and conventional synthesis procedures. In these reactions it is also possible to use variants which are known per se but are not mentioned herein.

For the purpose According to this invention, the chemical elements are in agreement with the Periodic Table of the Elements, CAS Version, Handbook of Chemistry and Physics, 67th Edition, 1986-87, Cover inside identified. Also for the purposes of this invention the term "hydrocarbon" is intended all permissible Compounds having at least one hydrogen and one carbon atom to include. In the broadest sense, the permissible hydrocarbons, acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic organic Compounds which may be substituted or unsubstituted.

Combinatorial libraries

The subject Reactions are easily suitable for the preparation of combinatorial libraries of compounds for the Screening of pharmaceutical, agrochemical or other biological or medicine-related activities or object-related qualities. A combinatorial library for the purposes of the present Invention is a mixture of chemically-related compounds, which together can be screened for a particular property; said library can be in solution or covalently bound to a solid support. The production reduced by many related compounds in a single reaction and simplifies the number of screening processes that are performed have to, strong. Screening for the appropriate biological, pharmaceutical, Agrochemical or physical property can be determined by means of conventional Procedure performed become.

The Diversity in a library can be a variety of different Layers are created. For example, those in a combinatorial Approach used substrate aryl groups with respect to the core aryl moiety be different, z. B., conceptual diversity of the ring structure, and / or can be varied with respect to the other substituents.

A variety of techniques are available in the art for preparing combinatorial libraries of small organic molecules. See, for example, Blondelle et al. (1995) Trends Anal. Chem. 14:83; the Affymax US Patents 5,359,115 and 5362899 ; the Ellman U.S. Patent 5,288,514 Still et al. PCT publication WO 94/08051 ; Chen et al. (1994) JACS 116: 2661; Kerr et al. (1993) JACS 115: 252; PCT publication WO 92/10092 . WO 93/09668 and WO 91/07087 ; and the Lerner et al. PCT publication WO 93/20242 ). Accordingly, a variety of libraries may be synthesized in the manner of about 16 to 1,000,000 or more diversomers and screened for a particular activity or property.

In an exemplary embodiment, a library of substituted diversomers may be prepared by the subject reaction described in Still et al. PCT Publication WO 94/08051 described technique are synthesized, for. By being bound to a polymer bead by a hydrolyzable or photolyzable group, e.g. B., located at one of the locations of the substrate. According to Still et al. Technique, the library is synthesized on a set of beads, each bead having a set of tags to identify the particular diver on the bead. In one embodiment particularly suitable for detecting enzyme inhibitors, the beads may be dispersed on a surface of a permeable membrane and the diversomers released from the beads by the lysis of the beaded linker. The diver of each bead will diffuse through the membrane into an assay zone in which it will interact with an enzyme assay. Detailed descriptions of a variety of combinatorial methodologies are provided below.

A. Direct characterization

One is a growing trend in the field of combinatorial chemistry the sensitivity of techniques such as mass spectroscopy (MS), which for Example can be used to subfemtomolate amounts of a compound and to characterize the chemical structure of the combinatorial Library selected Determine the connection directly. For example, where the library on an insoluble support matrix is provided individual populations of the compound first released from the carrier and be characterized by MS. In other embodiments, As part of MS sample preparation technology, MS techniques such as MALDI used to release a compound from the matrix, especially where a labile bond was originally used to bind the compound to the matrix. For example, an off a library selected globule be irradiated in a MALDI step to the diver of the Release matrix and ionize the diversomer for MS analysis.

B. Multipin Synthesis

The Libraries of the figurative Can process take the multipin library format. In short, Geysen and co-workers (Geysen et al. (1984) PNAS 81: 3998-4002) led a method for manufacturing of compound libraries by a parallel synthesis on polyacrylic acid-grafted Polyethylene pins, which are arranged in a microtiter plate format are a. The geyser technique can be used by thousands of compounds per week using the multipin procedure to synthesize and screen, and the bound compounds can for many Examinations are reused. Suitable linker residues may also be attached to the pins so that the compounds from the carriers after synthesis for evaluation purity and further evaluation can be split off (cf. Bray et al. (1990) Tetrahedron Lett 31: 5811-5814; Valerio et al. (1991) Anal Biochem 197: 168-177; Bray et al. (1991) Tetrahedron Lett 32: 6163-6166).

C. Separation-coupling-recombining

In yet another embodiment, a modified library of compounds may be provided on a set of beads utilizing the separation-coupling-recombination strategy (see, e.g., Houghten (1985), PNAS, 82: 5131-5135; U.S. Patents 4,631,211 ; 5440016 . 5480971 ). Briefly, as the name implies, at each synthesis step at which degeneration is introduced into the library, the beads are divided into separate groups equal to the number of different substituents to be added at a particular position in the library , the different substituents are coupled in separate reactions, and the beads are recombined to a pool for the next repetition.

In an embodiment can use the separation-coupling-recombine strategy using one to the so-called "tea bag" method analogous approach which were first developed by Houghten which is the synthesis of compounds on resins that are inside a porous polypropylene bag sealed (Houghten et al. (1986) PNAS 82: 5131-5135). Substituens are coupled to the compound-bearing resins, by placing the bags in appropriate reaction solutions while all joint steps, such as resin washing and protective elimination simultaneously be carried out in a reaction vessel. At the end of each synthesis contains each bag a single connection.

D) Combinatorial compounds by Light-directed, spatially-adaptable parallel chemical synthesis.

A combinatorial synthesis scheme in which the identity of a compound is given by its location on a synthetic substrate is called a spatially-addressable synthesis. In one embodiment, the combinatorial process is performed by controlling the addition of a chemical reagent to a specific site on a solid support (Dower et al (1991) Annu Rep Med Chem 26: 271-280; Fodor, SPA (1991) Science 251: 767 Pirrung et al. (192) U.S. Patent No. 5,143,854 ; Jacobs et al. (1994) Trends Biotechnol 12: 19-26). The spatial resolution of photolithography enables miniaturization. This technique can be accomplished by the use of protective / protective elimination reactions with photolabile protecting groups.

The most important steps of this technology are described in Gallop et al. (1994) J. Med. Chem. 37: 1233-1251 explained. A synthesis substrate is prepared for coupling through the covalent attachment of photolabile nitroveratryloxycarbonyl (NVOC) protected amino linkers or other photolabile linkers. Light is used to activate a specific region of the synthesis carrier for coupling. The removal of a photolabile protecting group by light (elimination of protection) leads to the activation of selected areas. Upon activation, a first set of amino acid analogs, each bearing a photolabile protecting group at its amino terminus, is exposed to the entire surface. The coupling occurs only in regions addressed by light in the previous step. The reaction is stopped, the plates are washed, and the substrate is again illuminated by a second mask, thereby activating a different region for reaction with a second protected building block. The pattern of the masks and the sequence of reactants define the products and their location. Since this method makes use of photolithography techniques, the number of compounds which can be synthesized is limited only by the number of sites of synthesis which can be addressed with adequate resolution. The position of each connection is known exactly; thus their interaction with other molecules can be assessed directly.

In In a light-directed chemical synthesis, the products depend on the exposure pattern and the nature of the addition of the reactants. By varying the lithographic pattern can many different sentences be synthesized on test compounds simultaneously; this characteristic leads to the development of many different masking strategies.

E) Coded combinatorial libraries

In a still further embodiment The object method uses a connection library that with equipped with a coded tagging system. A recent one Improvement in the identification of active compounds of combinatorial libraries uses chemical indexing systems, use the tags that the reaction steps a given globule has undergone, uniquely coded and whose structure is through conclusion wearing. In the concept, this approach mimics the phage display libraries in which the activity derived from expressed peptides, the structure of the active Peptides, however, derived from the corresponding genomic DNA sequence becomes. The first coding of synthetic combinatorial libraries used DNA as the code. A variety of other coding forms have been reported, including sequential coding Bio-oligomers (eg, oligonucleotides and peptides), and binary coding with additional non-sequencing- Tags.

1) Tagging with Sequenceable Bio-Oligomers

The principle of using oligonucleotides to encode combinatorial synthetic libraries has been described in 1992 (Brenner et al., (1992) PNAS 89: 5381-5383) and an example of such a library appeared the following year (Needles et al ) PNAS 90: 10700-10704). A combinatorial library of nominally 7 ⁷ (= 823,543) peptides, consisting of all combinations of Arg, Gln, Phe, Lys, Val, D-Val, and Thr (three-letter code for amino acids), each of which was encoded by a specific dinucleotide (TA, TC, CT, AT, TT, CA and AC, respectively) was prepared by a series of alternating rounds of peptide and oligonucleotide synthesis on a solid support. In this work, the amine-binding functionality on the bead was specifically differentiated toward peptide or oligonucleotide synthesis by simultaneously pre-incubating the beads with reagents that generate protected OH groups for oligonucleotide synthesis and protected NH ₂ groups for peptide synthesis (here in a ratio of 1:20). Each day, when complete, consists of 69-mers, of which 14 units carried the code. The bead-bound library was incubated with a fluorescently-labeled antibody and the bound antibody-bearing beads that fluoresced strongly were harvested by fluorescence activated cell sorting (FACS). The DNA tags were amplified by PCR and sequenced, and the predicted peptides were synthesized. Following such techniques, compound libraries can be derived for use in the subject method, where the oligonucleotide sequence of the tag identifies the combinatorial reactions of which a particular bead has been exposed and therefore provides the identity of the compound on the bead.

The use of oligonucleotide tags allows highly sensitive tag analysis. Nevertheless, the method requires the careful selection of orthogonal sets of protecting groups required for the alternate co-synthesis of the tag and the library members. Furthermore, the chemical lability of the tag, particularly the anomeric phosphate and sugar linkages, may limit the choice of reagents and conditions that can be used for the synthesis of non-oligomeric libraries. In preferred embodiments, the libraries use linkers that facilitate selective separation enable the Test Link Library member to investigate.

peptides are also called tagging molecules for combinatorial Libraries have been used. Two exemplary approaches are in the prior art, both of which have branched linkers on solid phases, on which coding and ligand strands alternatively be worked out. In the first approach (Kerr JM et al. (1993) J Am Chem Soc 115: 2529-2531) becomes orthogonality in the synthesis by applying an acid-labile protection for the coding strand and a base labile protection for the connection string.

at an alternative approach (Nikolaiev et al. (1993) Pept Res. 6: 161-170) branched linkers used, so that both, the coding unit and the test compound to the same functional group on the Resin can be attached. In one embodiment, a fissile Be placed left between the branching point and the bead, so that the split is the molecule which contains both the code and the compound (Ptek et al. (1991) Tetrahedron Lett 32: 3891-3894). In another, embodiment The cleavable linker can be placed so that the test compound leaving behind of the code selectively from the bead can be separated. This last construct is especially valuable since it screening the test compound without potential impairment the coding groups. Examples in the prior art of independent Cleavage and sequencing of peptide library members and their corresponding tags have confirmed that the tags are the peptide structure accurately predict.

2) Non-sequenceable tagging: Binary coding

An alternative coding format of the test compound library uses a set of non-sequencable electrophoric tagging molecules that are used as a binary code (Ohlmeyer et al. (1193) PNAS 90: 10922-10926). Exemplary tags are haloaromatic alkyl ethers which are detectable as their trimethylsylyl ethers at lower than femtomolar concentrations by electron capture gas chromatography (ECGC). Variations in the length of the alkyl chain, as well as the nature and position of the aromatic halide substituents, enable the synthesis of at least 40 such tags, which in principle can encode 2 ⁴⁰ (eg upwards of 10 ¹² ) different molecules. In the original report (Ohlmeyer et al., Supra), the tags were attached to about 1% of the available amine groups of the peptide library via a photocleavable o-nitrobenzyl linker. This approach is useful when preparing combinatorial libraries of peptide-like or other amine-containing molecules. However, a more versatile system has been developed which enables the coding of essentially any combinatorial library. Here, the compound would be linked to the solid support via a photocleavable linker and the tag would be linked to the bead matrix by a catechol ether linker via a carbene insertion (Nestler et al., (1994) J Org Chem 59: 4723-4724). , This orthogonal joining strategy allows the selective separation of library members for solution testing and subsequent decoding by ECGC after oxidative separation of the tag sets.

Even though several amide-linked libraries in the prior art binary coding with the electrophores tags attached to the amino groups Use provides direct binding of these tags to the bead matrix a much greater versatility in the structures used in coded combinatorial libraries can be produced. Bound in this way, the tags and their linkers are almost as unreactive as the bead matrix itself. Two binary-coded Combinatorial libraries have been reported in which the electrophoresis Tags are bound directly to the solid phase (Ohlmeyer et al. (1995) PNAS 92: 6027-6031) and provide instructions for the preparation of the subject compound library. Both Libraries were constructed using an orthogonal connection strategy in which the library member passes to the solid support a photolabile linker was bound and the tags were through a linker which is cleavable only by strong oxidation bound. Since the library members are repeating part of the solid support Photo-eluted, can the library members are used in many investigations. The successive photoelution allows also a very high throughput repetitive screenings strategy: firstly several beads placed in 96-well microtiter plates; second are the connections partially separated and transferred to assay plates; third identified a metal binding assay the active chambers; Fourth the corresponding beads individually rearranged into new microtiter plates; fifth individual active compounds identified; and sixth, the Structure decoded.

Automation of solid-phase oligosaccharide synthesis

Of the Access to defined sequences of polypeptides and nucleic acids by automated synthesis had a significant impact on the Research in a variety of fields including these molecules. Important Technologies like PCR became possible after a reliable one Access to short DNA strands Became routine. The field of glycobiology has been affected by the difficulties suffered in synthesizing complex oligosaccharides. Ideally, you could researchers who are interested in phenomena which contain complex glycoconjugates are automated Support synthesis, around the necessary molecules manufacture.

In a general application, the device of the present Invention for uses the preparation of oligosaccharides or mixtures thereof which for Structure-function analysis of oligosaccharides can be used. For example, the / the Oligosaccharide (s) of interest antigenic oligosaccharides, oligosaccharides, which are involved in protein carbohydrate detection processes, or a be antibiotic oligosaccharide. Typically it is for example desirable in structure-function studies, the impact on the activity of one of a plurality of sugar substituents on one or more of several selected To determine remaining positions. In another general application For example, the device is used to sequence random oligosaccharides for selecting Oligosaccharides with desired Activity, typically binding activity to a known receptor, such as to produce an antibody.

While recent advances in the development of solid phase oligosaccharide synthesis have been achieved, no attempts have been reported to automate solid phase oligosaccharide synthesis. Encouraged by the success of our attempts to synthesize oligosaccharides on a solid support, we began assembling carbohydrates in a synthesizer. We assembled a pentasaccharide in 8.5 hours with a total yield of 75% in a fully automated manner. An ABI 393B peptide synthesizer equipped with a custom-made reaction vessel was used to take up the solutions of mannosetrichloroacetimidate donor, activator (TMSOTf), and sodium methoxide in methanol as a deblocking solution, methylene chloride, THF, and methanol were used as wash solvents while all reagents remained under an inert gas atmosphere. The coupling cycle was programmed to deliver five equivalents of donor and catalytic amounts of activator to the resin, followed by a 20 minute wait. After the washing step, glycosylation was repeated to ensure high yields. After rinsing, a 20 minute deprotection revealed the next acceptor site. Repetition of this protocol gave this desired pentasaccharide in excellent stepwise coupling yields (> 98%) and purity. The HMQC Spektra of carrier-bound crude pentasaccharide prepared by fully automated synthesis in 500 minutes is compared to the solution HMQC spectrum of pure reference pentasaccharide n-pentenyl glycoside in solution. Please refer 17 , Although the linewidth of the HR-MAS spectrum widened due to the polymer, it provides convincing evidence that the desired product was observed to be the major compound after 10 synthetic steps. We have begun to evaluate the use of other glycosyl donors in combination with different carriers and shorter coupling protocols to assemble the larger oligosaccharides of biological significance.

Protocols for automated solid-phase synthesis of oligosaccharides

reactions on the solid support are especially good for the automation. In a preparatory work, we have a trimannoside with an overall yield of 91% on a peptide synthesizer synthesized, which regards the solvent and reagent supply was adjusted.

Solid phase linker

One crucial element of any solid-phase synthesis strategy is the Choice of a suitable linker group, the first sugar to the Solid support binds (Table 1). For an overview at linkers for the organic solid phase synthesis see: F. Guillier, D. Orain, M. Bradley, Chem. Rev., 100 (2000), 2091. The linker must be opposite the Glycosylierungsbedingungen as well as the protective elimination conditions be inert. Of equal importance is the need for one High yield cleavage to complete the synthesis. Several acidic and broomlabile linkers, similar to those used in peptide and oligonucleotide synthesis be, are for the oligosaccharide synthesis has been modified.

Table 1: Linker and cleavage method for solid phase oligosaccharide synthesis

silylether become the most common for the Monomer bond to the polymer support under the donor-bonded Used paradigm. Linkers such as diisopropylphenylsilyl ether 1 across from mildly acidic and strongly basic reaction conditions stable and have come in handy proved when glycosyl trichloroacetimidate, Glycale, Glycosylfluoride and glycosyl sulfoxides are used. The release from the polymer carrier is achieved by treatment with fluoride sources containing the free Oligosaccharide which is unprotected at the original binding site, supplies. Due to the frequent Use of Silyl Ether as Temporary Protecting Groups in Oligosaccharide Synthesis leads the Incorporation of a silyl linker often leads to significant challenges, when differentiated between the remaining hydroxyl groups should.

A Class of photolabile linkers has been developed to use to bypass the use of silyl ether as a linker and to the use of temporary silyl protecting groups enable. U. Zehavi, A. Patchomik, J. Am. Chem. Soc., 95 (1973), 5673; R. Rodebaugh, B. Fraser-Reid, H.M. Geysen, Tetrahedron Lett. 38 (1997), 7653. Photolabile linkers, such as 2, often involve the use of o-nitrobenzyl ether groups. This functional group is opposite to one Series of conditions stable, which is cleavage of polymer carrier however, often very slowly and the yields vary. caging Linkers still offer a different degree of orthogonality, which useful is when a synthesis is drafted.

A unique linker strategy for oligosaccharide synthesis involves attachment of the first sugar to the polymer support via a thioglycoside. As discussed above, thioglycosides are moderately stable to acid and inert under basic conditions. Thioglycosides, such as 3, are cleaved from the polymer support by the addition of thiophilic reagents such as N-bromosuccinimide or Hg (O ₂ CCF ₃ ) ₂ in the presence of an alcohol or water to give either acetal or hemiacetal products. The obvious limitations of this linker are incompatibility with thioglycoside donors and limited stability over treatment with acid activators. A mixture of anomers is also possible and control of the anomeric ratio is difficult, as the cleavage process occurs at the reducing end.

First recently are linkers, similar to 4, which exploited olefin metathesis as the cleavage method, been developed. Olefin linkers are resistant to acidic and basic reaction conditions stable and the double bond acts as an approach to the final cleavage process. This option is attractive because the linker functionality is under the for usually used coupling and deprotection conditions inert is and the final one Cleavage step is compatible with numerous protecting groups. From Meaning is that the cleavage product contains an olefin, which additional functional group manipulations allows.

Analogous to the solution phase oligosaccharide synthesis does not have a single glycosylation or linker strategy among the variety of available Solid phase techniques awarded. The richness of linker strategies offers a useful Flexibility, when a synthesis is planned. It is anticipated that the Development of novel linkers and cleavage strategies the synthesis of increasingly complex carbohydrates using solid-phase methods allows.

coupling cycle

The Solid phase coupling conditions developed by us were used in the Assembly of Oligosaccharides in an Automated Way and Way applied. Many glycosylation reactions can be included Room temperature performed Be and include reagents that are completely soluble and prolonged Storage at room temperature are stable. We initially used glycosyl trichloroacetimidate donors as a monosaccharide unit, because these units can be coupled at room temperature. ester and silyl protecting groups were used as temporary masking groups, their removal is done in high yield and fast.

The heptamannoside prepared by us served as the first test sequence for the Coupling cycle. Because no monitoring carried out was, had to complete Coupling and protection elimination are ensured. In one typical coupling cycle were five equivalents of glycosyl donor and the activator TMSOTf were supplied to the resin. After an hour was the resin rinsed, and the coupling step is repeated. After rinsing the resin and several Washing steps, sodium methoxide in methanol was added to the resin and for one hour react. The next Coupling cycle started after another washing step. In connection At the conclusion of the synthesis, the resin became a cross-metathesis subjected to ethylene to form the finished oligosaccharide in the form of a to remove n-pentylglycoside. The success of automated synthesis was added by analytical HPLC by comparing the amount of product the sum of by-products (mainly deletion sequences) rated.

introduction a capping step

deletion sequences, where only one sugar unit (n - 1) is missing are the most difficult from the desired Separate product and arise from incomplete coupling steps, while any coupling cycle of the sequence. The oligosaccharide chains, the while can not couple a cycle during the following extension steps successfully glycosylated. It can therefore be a serious one Cleaning problem at the end of the synthesis exist. To the extension Failed sequences can avoid a capping step (i.e., a blocking step) are introduced into the coupling cycle. After each complete coupling can be a highly reactive blocking group used to cover all free hydroxyl acceptors. For example, benzyl trichloroacetimidate may be used as a capping reagent (activated with TMSOTf) can be used to position benzyl ethers in positions which were not glycosylated and around these during the to render the whole synthesis unreactive. Using this direct Capping step, it is expected that the cleaning of the finished Oligosaccharide product is greatly simplified since the presence its (n-1) deletion sequences will be minimized.

Automated solid-phase synthesis of oligosaccharides

The transmission of information at the molecular level is a central process in life, which in the cellular system is mainly based on three major repeating biopolymers: proteins, nucleic acids and carbohydrates (see 4 ). While the importance of proteins and nucleic acids has long been recognized in biology, the understanding of oligosaccharides and glycoconjugates in nature is still in its infancy. RA Dwek, Chem. Rev. 96, 683 (1996). Cell surface glycoconjugates are involved in signal transduction pathways and cellular recognition processes and have been implicated in many disease states. S. Hakomori, Adv. Cancer Res. 52, 257 (1989).

One major barrier in the fast-washing field of molecular glycobiology is the lack of pure, structurally-defined carbohydrates and glycoconjugates. These biomolecules are often found in nature in low concentrations and in microheterogeneous form, which makes their identification and isolation extremely complicated. The procurement of sufficient quantities of defined oligosaccharides, which are required for detailed biophysical and biochemical studies, therefore relies on efficient synthetic procedures. While much progress has been made in oligosaccharide synthesis, the construction of complex carbohydrates remains time consuming and is performed by a small number of specialized laboratories. GJ Bonns, ed., Carbohydrate Chemistry (Blackie Pub Lishers, London, 1998).

Historical seen was access to structurally-defined complex carbohydrates very expensive. recent Advances in solid-phase synthesis have led to the construction of complex oligosaccharides made less tedious, it is, however still a high level technical expertise needed to obtain the desired structures. Here we describe an automated chemical synthesis of several oligosaccharides on a solid phase synthesizer. By the use of glycosyl phosphate building blocks and a novel functionalized resin, a branched dodecasaccharide was synthesized. The target oligosaccharide was easily separated after cleavage Solid support receive. Access to complex oligosaccharides has now also for non-experts, in a way possible, in a manner such as the construction of oligopeptides and Oligonucleotides.

At long last becomes a general, automated process for oligosaccharide composition fast production of interesting structures, even for one Non-specialists allow. Oligonucleotides (M.H. Caruthers, Science, 230, 281 (1985)) and Oligopeptides (E. Atherton, R.C. Sheppard, Solid phase peptide synthesis: a practical approach, (Oxford University Press, Oxford, 1989)) now routinely on an efficient way using solid phase strategies produced by automated synthesizers. The effect an automated synthesizer in the field of glycobiology can easily visualized when the significant influence which the peptide and Nucleotide devices on the biochemistry of these biopolymers had considered becomes.

Automated Oligosaccharide Synthesizers

We have the first automated solid-phase oligosaccharide synthesizer developed. The solid phase paradigm is particularly well suited to automation of synthetic processes. The repetitive nature of glycosylation and protection elimination can be easily incorporated into a coupling cycle become. A surplus Reagents are used to complete the reactions respectively, while Resin washes (through the use of solvents) any soluble contaminants away. Only a single cleaning step is required after the sugar has been released from the solid carrier.

Under Considering the advantages of solid support synthesis, considered we have several key questions for development an automated oligosaccharide synthesizer: a) the design an overall synthetic strategy, where either the reducing or the non-reducing end of the growing carbohydrate chain to the carrier (See Y. Ito, S. Manabe, Curr. Opin. Chem. Biol. 701 (1998)); b) Selection of a polymer and linkers that are opposite to all Reaction conditions during the synthesis are inert, but are cleaved efficiently if desired; c) protecting group strategy consistent with the complexity of the target oligosaccharide; d) stereospecific and high yield glycosylation reactions; and e) an instrument capable of repeating, chemical Performing operations at different temperatures.

Furthermore, requires the moisture sensitivity of the glycosylation reaction a system kept continuously under an inert gas atmosphere can be. In addition, since many glycosylation reactions require lower temperatures, can a glass reaction vessel, which surrounded by a second cavity that controls the circulation of a refrigerant allows be used. The catalytic use of activators, such as Trimethylsilyltrifluoromethanesulfonate requires very accurate measurement and feeder of these reagents.

Instead of a new device to design, we preferred an existing device, which is used in automated peptide synthesis to revise. The peptide synthesizer Model 433A, available from Applied Biosystems Inc. available is, was for modified our purposes. Several modifications were necessary before the commercially available Peptide synthesizer for the carbohydrate synthesis could be used. The device was equipped with several reagent bottles, some for washing and others for the glycosylation and protection elimination reactions. Small cartridges, which contain the donor species can be manually synthesized loaded into the synthesizer. One cycle has been programmed to perform all steps without any operator intervention. The Device controlled the delivery of all reagents and Donator species to the reaction vessel and the mixing of the vessel contents.

Referring to 5 , Will be an embodiment of an automated oligosaccharide synthesis tisierers 200 shown constructed according to the invention. The shown automated oligosaccharide synthesizer 200 consists of a reaction vessel 210 , two donor vessels 220a -B containing monosaccharide donor solutions, an activator vessel 230 containing an activating reagent solution, a deblocking vessel 240 containing a deblocking reagent solution, a blocking vessel 250 containing a blocking reagent solution, three solvent vessels 260a -C, which contain solvent solutions, a solution transfer system 270 , a temperature control unit 290 for the regulation of the temperature of the reaction vessel 210 (keep the temperature of the reaction vessel 210 at a desired temperature (s)), and a computer 280 , which can be programmed to the solution transfer system 270 and the temperature control unit 290 to automatically control.

The donor vessels 220a -B can take up any suitable glycosyl donor solution, such as a glycosyl trichloroacetimidate or a glycosyl phosphate. The activator vessel 230 can take up any suitable activating reagent solution. In general, Lewis acids have been shown to contribute to the formation, ie, synthesis, of oligosaccharides and therefore can be used as a suitable activator. Thus, the activator vessel 230 a solution comprising a silyl trifluoromethanesulfonate or, alternatively, may contain a solution comprising trimethylsilyl trifluoromethanesulfonate. The deblocking vessel 240 may include any suitable deblocking reagent solution, such as a solution containing sodium methoxide or hydrazine. The blocking vessel 250 may contain any suitable blocking reagent solution, such as a solution containing benzyl trichloroacetimidate or a carboxylic acid. One such suitable carboxylic acid is levulinic acid. The variety of solvent vessels 260a -C may contain any suitable solvent solution, inter alia, such as dichloromethane, THF, and methanol.

The solution transfer system 270 must be able to transfer the donor, the activation, the deblocking and the solvent solutions from their respective vessels into the reaction vessel 210 to convict. Due to the moisture sensitivity of certain glycosylation reactions, the solution transfer system should be able to handle the reagents of the device 220 under an inert gas atmosphere, ie to keep the reagents under pressure. Those skilled in the art will recognize that such solution transfer systems 270 are commercially available and readily available; for example, the system of Applied Biosystems Inc, Model 443 A peptide synthesizer. The im U.S. Patent 5,186,898 also described solution transfer system is also a system which would be suitable for the synthesis of oligosaccharides according to the present invention.

The computer 280 Any suitable computing device can control the workflows of the solution transfer system 270 and the temperature control unit 290 be such as a personal computer or workstation. The computer 280 can be preprogrammed so that it automatically the workflows of the solution transfer system 270 controlled; The coupling, washing, protecting / capping (ie blocking), and protection elimination cycles can be done in the computer for a given protocol 280 be preprogrammed. In this way, the automated solid-phase synthesis of oligosaccharides can be controlled and achieved. In addition, the computer can 280 a device which is separate from the solution transfer system 270 is, or the computer 280 can enter the solution transfer system 270 be installed. If the computer 280 and the solution transfer system 270 are separate devices, then a suitable data communication path, such as a communications port / cable or IR data link, must be present between the two devices. Similarly, if an automatic temperature control of the reaction vessel 210 is desired, then the computer can 280 be preprogrammed with the desired temperature protocol, so that the operations of the temperature control unit 290 to be controlled. For the automatic control of the temperature control unit 290 over the computer 280 , the computer needs 280 in data communication with the temperature control unit 290 stand.

The temperature control unit 290 may be any suitable device which is capable of controlling the temperature of the reaction vessel and maintaining it at the desired temperatures. Several of the external refrigerated circulators available from Julabo USA Inc, Allentown, PA can be considered as an acceptable temperature controller 290 be used. To achieve the automated solid-phase synthesis of many different types of oligosaccharides, temperature control should be included 290 to be able to measure the temperature of the reaction vessel 210 at a set temperature of between -25C and +40, and preferably at a set temperature of between -80C and + 60C. The coolant of the temperature control unit 290 can around the reaction vessel 210 via a sleeve (not shown) which can cover the reaction vessel and which with the temperature control unit 290 connected via an input line and output line, are circulated. Alternatively, the reaction vessel 210 have a double-walled structure, wherein an external cavity of the double-walled structure the Coolant of the temperature control unit receives. The temperature of the reaction vessel 210 can by pre-programming the temperature control unit 290 be adjusted to the desired temperature and then allow the coolant to the reaction vessel 210 for a predetermined "cold-penetrating" period, such as 5 minutes, to circulate around. Alternatively, the temperature control unit can be placed on the wall of the reaction vessel 210 have placed temperature sensor, allowing real-time temperature measurement of the actual reaction vessel 210 Cavity are obtained, ie where the automated synthesis of oligosaccharides takes place. Thus, the temperature sensor may provide feedback data to the temperature control unit 290 provide so that the actual temperature of the reaction vessel 210 can be maintained more accurately.

Referring to 7 is an embodiment of a double-walled reaction vessel 210 (shown in section) constructed in accordance with the invention. The double-walled structure of the reaction vessel 210 forms two cavities: the first cavity 210a houses the synthesis of oligosaccharides; the second cavity 210b takes the coolant of the temperature control unit 290 on. The coolant of the temperature control unit 290 circulates over the pipelines 291 and 292 through the second cavity 210b , These pipelines 291 and 292 can be made of any suitable material, such as rubberized materials or metallic materials. The pipelines 291 and 292 can be attached to the two openings in the outer surface of the jacketed reaction vessel 210 via mechanical clamps, tapes, adhesives, epoxies, etc. The double-walled, cooled reaction vessel 210 can be made of glass or any other suitable material, such as titanium.

The different solutions are in the cavity 210a via the solution transfer system 270 introduced ( 5 ). Likewise, these solutions may be out of the cavity 210a By the operation of the solution transfer system 270 - be forced out by the introduction of an additional solution or by the introduction of a compressed inert gas (to be collected as waste). Prior to operation of the synthesizer, insoluble resin beads may be used 214 in the cavity 210a of the reaction vessel 210 to be placed. The resin beads 214 may consist of an octenediol functionalized resin and may further include an organic linker to the resin bead 214 having bound glycosyl acceptor (not shown). The organic linker may consist of a glycosyl phosphate. The solutions can be the cavity 210a over a porous glass frit 216 leave. The glass frit 216 allows the passage of the solution, but keeps the resin beads 214 and more importantly, the oligosaccharides formed thereon within the cavity 210a of the reaction vessel 210 back.

explanation

The now generally described invention is by reference to the following examples better understandable, which for illustrative purposes only certain aspects and embodiments of the present invention, and not intended are to limit the invention.

example 1

Automated Solid Phase Oligosaccharide Synthesis - General experimental methods

General procedure

All chemicals used were reagent grade and were used as supplied except where indicated. Dichloromethane (CH ₂ Cl ₂ ) used for wash cycles was purchased from Mallinekrodt (HPLC grade) and used without further purification. The dichloromethane (CH ₂ Cl ₂ ) used for the reagent preparation was purchased from JT Baker (Cycletainer ^™ ) and passed through a neutral alumina column prior to use. Tetrahydrofuran (THF) was purchased from JT Baker (Cycletainer ^™ ) and passed through a neutral alumina column prior to use. Pyridine was refluxed over calcium hydride and distilled before use. Trimethylsilyl trifluoromethanesulfonate (TMSOTf) was purchased from Acros Chemicals. Sodium methoxide (25% w / v in MeOH), glacial acetic acid (AcOH) and hyracine acetate (98%) were purchased from Aldrich Chemicals. Analytical thin layer chromatography was performed on E. Merck silica gel 60 F ₂₅₄ plates (0.25 mm). Compounds were visualized by immersing the plates in a cerium sulfate-ammonium molybdate solution followed by heating. Liquid column chromatography was performed using forced flow of the indicated solvent on a Silicycle 230-400 mesh (60Å pore diameter) silica gel. H NMR spectra were obtained on a Varian VXR-500 (500 MHz) spectrometer and are reported in parts per million ppm with respect to CHCl ₃ (7.27 ppm). Coupling constants (J) are given in Hertz. C NMR spectra were with obtained from a Varian VXR-500 spectrometer (125 MHz) and are reported in terms of CDCl ₃ (77.23 ppm) as an internal reference. Polymer-bound compounds were analyzed by HR-MAS NMR using the following conditions: All spectra were run on a Bruker DRX-600 spectrometer operating at 600 MHz (H) equipped with a 4 mm Bruker CCA HR-MAS probe , analyzed. Samples (10 mg at 0.45-0.55 mmol g ^-1 ) were loaded into a ceramic rotor, suspended in 30-100 LCDCL ₃ and rotated at the magic angle at 3.0 KHz. 1D NMR analysis was performed using a 1D spin echo: 32 scans, 2 s relaxation time, 3 minutes total experiment time. TOCSY data were obtained using: mlevtp pulse sequence, 80 milliseconds mixing time, 1 second relaxation delay, 16 scans per free induction decay (FID), 400 FIDs, 2048 points per FID, 2.2 hours total experiment time HMQC experiments were performed using: invbtp Pulse sequence (HMQC with BIRD sequence), 1.3 second relaxation delay, 96 scans per FID, 256 FIDs, 2048 points per FID, 13.5 hours total experiment time. MALDI-TOF mass spectrometry was performed on a PE Biosystems Voyager System 102 as follows: Al 1 aliquot of the matrix solution [10 mg / mL 2,5-dihydroxybenzoic acid (DHB) in THF] was spotted onto the sample holder and allowed to dry. Addition of an aliquot of the oligosaccharide solution (5 mg / ml EtOAc) was simultaneously spotted onto the matrix, dried, and analyzed in positive ion mode. HPLC analysis was performed on a Waters Model 600E multisolvent dispensing system using analytical (Nova- ^Pak® , 60 Å, 4 m, 3.6 x 150 mm) and preparative (Nova- ^Pak® , 60 Å, 6 m, 7.8 × 300 mm) silica columns.

General Method A. Automated Synthes of - (1 → 2) -mannosides:

Octene diol functionalized resin 1 (25 mol, 83 mg, 0.30 mmol / g loading) was charged to a reaction vessel and inserted into the oligosynthesizer. The resin was purified using donor 2 (10 equiv, 0.25 mmol, 160 mg), which was dissolved in CH ₂ CL ₂ (4 mL) and TMSOTF (0.5 equiv, 1 mL, 0.0125 M TMSOTf in CH ₂ Cl ₂ ) was added, glycosylated. The mixing of the suspension was carried out (10 sec vertex, 50 sec rest) for 30 minutes. The resin was then washed with CH ₂ Cl ₂ (6 x 4 ml each) and glycosylated a second time. After the second glycosylation was stopped, the resin was washed with CH ₂ Cl ₂ (6 x 4 mL each) and with 1: 9 MeOH: CH ₂ Cl ₂ (4 x 4 mL, each). The deprotection of the acetyl ester was carried out by treating the glycosylated resin with sodium methoxide (10 equiv, 0.5 ml, 0.5 M NaOMe in MeOH) in CH ₂ Cl ₂ (5 ml) for 30 minutes. The resin was then washed with 1: 9 MeOH: CH ₂ Cl ₂ (1 x 4 mL) and exposed a second time to the deprotection conditions for 30 minutes. Removal of all soluble impurities was accomplished by washing the resin with 1: 9 MeOH: CH ₂ Cl ₂ (4 x 4 mL, each), 0.2 M AcOH in THF (4 x 4 mL each), THF (4 x 4 mL each ), and CH ₂ Cl ₂ (6 x 4 ml each). The deprotected polymer-bound C2-OH mannoside was then extended by repeating the above glycosylation / deprotection protocol. The terminal glycoside was not deprotected, thereby simplifying NMR analysis of the cleaved product.

General Method B. Oligosaccharide cleavage from the polymer carrier

The glycosylated resin (25 mol) was dried in vacuo over phosphorus pentoxide for 12 hours and transferred to a 10 ml bottle. The bottle was purged with ethylene and Grubbs catalyst (bis (tricyclohexylphosphine) benzylidine-ruthenium (IV) dichloride, 4.1 mg, 0.005 mmol, 20 mol%) was added. The reaction mixture was diluted with CH ₂ Cl ₂ (3 mL) and stirred under about 1 bar (1 atm) of ethylene for 36 hours. Triethylamine (111 ml, 0.80 mmol, 160 equiv.) And trishydroxymethylphosphine (50 mg, 0.40 mmol, 80 equiv.) Were added and the resulting solution was stirred at room temperature for 1 hour. HD Maynard, RH Grubbs Tetrahedron Lett. 40, 4137 (1999). The pale yellow reaction mixture was diluted with CH ₂ Cl ₂ (25 mL) and washed with water (3 x 25 mL), saturated aqueous NaHCO ₃ (3 x 25 mL), and brine (3 x 25 mL). The aqueous phase was extracted with CH ₂ Cl ₂ (25 mL) and the combined organic phases were dried over Na ₂ SO ₄ , filtered and concentrated. The resulting oligosaccharides were purified by either flash column chromatography on silica gel or high performance liquid chromatography (HPLC).

Pentamannoside 3.

Produced by running of general procedure A. The H-NMR spectrum of pentamer 3 was in in every way with an authentic sample that synthesizes in the first place became identical. R.B. Andrade, O.J. Plante, L.G. Melean, P. H. Seeberger, Org. Lett. 1, 1811 (1999). HR-MAS Spektra of resin-bound Pentamers was recorded using the HMQC and TOCSY methods and are inserted below. Alles five characteristic anomeric proton resonances were observed in the HR-MAS TOCSY spectrum (4.8-5.5 ppm), including of two anomeric protons that overlap in the HMQC spectrum.

Heptamannoside 4.

Produced by running general procedure A. The spectral data of heptamer 4 were in in every way with an authentic sample synthesized by the leader became identical. R.B. Andrade, O.J. Plante, L.G. Melcan, P. H. Seeberger, Org. Lett. 1, 1811 (1999). An H-NMR of by preparative HPLC purified pure 4 of heptamannoside synthesis is inserted below.

analytical HPLC chromatogram of heptamannoside 4 synthesis, followed by the resin splitting. flow rate = 1 ml / min, 20 → 30% EtOAc / hexanes (20 minutes): 7.8 minutes heptamannoside 4, 6.2 minutes logo CNRS logo INIST Hexamer, 5.4 minutes = pentamer.

Decamannoside 5

Prepared by carrying out general procedure A. The decamer was purified by preparative HPLC and characterized by H-NMR and MALDI-TOF. Characteristic anomeric resonances in the ¹ H-NMR spectrum (4.9-5.4 ppm) confirmed the decamer structure. A single acetate resonance (2.1 ppm) and n-pentenyl resonance, along with mass spectrometric data (calculated M ⁺ + Na (4473.6) found MALDI-TOF (DHB, THF) (M ⁺ + Na (4474.0) ) uniquely identify the decamer 5.

analytical HPLC chromatogram of decamannoside 5 synthesis, followed by the resin splitting. flow rate = 1 ml / min, 20 → 25% EtOAc / hexanes (20 minutes): 5.7 minutes = decamannoside 5, 5.0 minutes Nonamer, 4.4 minutes = Octamer.

General Method C. Automated Synthesis of Phytoalexin Inducers - Glucan Oligosaccharides:

Octene diol-functionalized resin 1 (25 mol, 83 mg, 0.30 mmol / g loading) was loaded into a reaction vessel equipped with a cooling jacket and inserted into a modified ABI-433A peptide synthesizer. The resin was purified using donor 8 or 9 (5 equiv., 0.125 mmol, 90 mg, and 146 mg, respectively) dissolved in CH ₂ CL ₂ (4 mL) and TMSOTF (5 equiv, 1 mL, 0.0125 M TMSOTf in CH ₂ Cl ₂ ) at -15 ° C, glycosylated. Mixing of the suspension was carried out (10 sec vertex, 50 sec rest) for 15 minutes. The resin was then washed with CH ₂ Cl ₂ (6 x 4 ml each) and glycosylated a second time. After the second glycosylation was stopped, the resin was washed with 1: 9 MeOH: CH ₂ Cl ₂ (4 x 4 mL, each), THF (4 x 4 mL) and pyridine: acetic acid (3: 2, 3 x 4 mL) and heated to 15 ° C. The deprotection of the levulinoyl ester was carried out by treating the glycosylated resin with hydrazine acetate (40 equiv, 4 ml, 0.25 MN ₂ H ₄ -HOAc in pyridine: acetic acid 3: 2) for 15 minutes. The resin was exposed a second time to the deprotection conditions for 15 minutes. Removal of all soluble impurities was accomplished by washing the resin with pyridine: acetic acid (3: 2, 3 x 4 mL), 0.2 M AcOH in THF (4 x 4 mL each), THF (4 x 4 mL each), and CH ₂ Cl ₂ (6 x 4 ml each). The deprotected polymer-bound C6-OH glucoside was then extended by repeating the above glycosylation / deprotection protocols and using alternating donors 8 and 9. The terminal glycoside was not deprotected, thereby simplifying the NMR analysis of the products released by use of Procedure B.

Phytoalexin-Inducer Hexasaccharide 10

Prepared by carrying out General Method C. The hexamer was purified by preparative HPLC and characterized by ¹ H-NMR and MALDI-TOF. The characteristic t-butyl (1.2 ppm), n-pentenyl (5.7 ppm) and levulinoyl (2,2, 2,5, 2,7) resonances together with mass spectrometric data (calculated M ⁺ + Na (2776, 8) found MALDI-TOF (DHB, THF) M ⁺ + Na (2778.0)) clearly identify the hexamer 10.

analytical HPLC chromatogram of the PE hexasaccharide 10 synthesis, subsequently and the resin splitting. flow rate = 1 ml / min, 17% EtOAc / hexanes: 4.75 minutes hexamer.

Phytoalexin Inductor Dodecasaccharide 7

Prepared by carrying out General Method C. The dodecamer was purified by preparative HPLC and characterized by ¹ H-NMR, ¹³ C-NMR and MALDI-TOF. The characteristic t-butyl (1.2 ppm), n-pentenyl (5.7 ppm) and levulinoyl (2,2, 2,5, 2,7) resonances together with mass spectrometric data (calculated M ⁺ + Na (5345, 5) found MALDI-TOF (DHB, THF) M ⁺ + Na (5346.2)) unambiguously identified dodecamer 7.

analytical HPLC chromatogram of PE dodecasaccharide 7 synthesis, subsequently and the resin splitting. flow rate = 1 ml / min, 20 → 25% EtOAc / hexanes: 6.7 minutes dodecamer.

Example 2

• Glycosylierungsbedingungen: 25 μmol Maßstab; 25 μmol Harz (83 mg, 0,30 mmol/g Beladung); 10 Äquiv. Donator 2 (160 mg); 0,5 Äquiv. TMSOTf (1 ml, 0,0125 M TMSOTf in CH₂Cl₂) zweimal für jeweils 30 Minuten wiederholt.
• Schutz-eliminierungsbedingungen: 25 μmol Maßstab; 10 Äquiv. NaOMe (0,5 ml, 0,5 M NaOMe in MeOH) in 5 ml CH₂Cl₂, zweimal für jeweils 30 Minuten wiederholt.

Using the modified peptide synthesizer, a systematic study of the variables involved in automated solid-phase oligosaccharide synthesis was undertaken. We selected the "acceptor-bound" strategy for solid-phase oligosaccharide synthesis. B. Merrifield, J. Am. Chem. Soc. 85, 2149 (1963). In this method, the reactive glycosylating agent is supplied in solution while the nucleophilic Akzepor hydroxyl group is exposed to the solid support. The productive coupling events result in carrier-bound oligosaccharides which are easily purified by washing the soluble by-products through a filter. Removal of a temporary protecting group on the newly formed saccharide moiety released another hydroxyl group, which allowed the coupling cycle to continue. The synthesis of biologically important mannosides was the focus of significant research; therefore, these molecules (Scheme 1, 3-5) were chosen to establish an efficient automated cycle. Trichloroacetimidate donor 2 was chosen as the donor assembly since it can be prepared on a multigram scale and activated at room temperature. See J. Rademann, RR Schmidt, J. Org. Chem. 62, 3989 (1997) and references therein. Activation of 2 was performed under acidic conditions using the Lewis acid trimethylsilyl trifluoromethanesulfonate (TMSOTf). Removal of the acetyl ester protecting group was achieved under basic conditions with sodium methoxide. Scheme 1: Automated oligosaccharide synthesis using trichloroacetimidates

• Glycosylation conditions: 25 μmol scale; 25 μmol resin (83 mg, 0.30 mmol / g loading); 10 equiv. Donor 2 (160 mg); 0.5 equiv. Repeat TMSOTf (1 mL, 0.0125 M TMSOTf in CH ₂ Cl ₂ ) twice for 30 minutes each.
• Protection elimination conditions: 25 μmol scale; 10 equiv. NaOMe (0.5 mL, 0.5 M NaOMe in MeOH) in 5 mL CH ₂ Cl ₂ , repeated twice for 30 minutes each.

To allow the use of acidic and basic reaction conditions in the coupling cycle, a polymer support and linker for compatibility were examined. RB Andrade, OJ Plante, LG Melean, PH Seeberger, Org. Lett. 1, 1811 (1999). A variety of commercially available polymer Trä gladly was examined. Merrifield's resin (1% cross-linked polystyrene) and polystyrene-based agarose showed excellent properties throughout the coupling cycle. Our preliminary work showed that the olefinic linker 1 was stable to the coupling cycle conditions, while it was readily cleaved from the solid support at the end of the synthesis by olefin cross-metathesis. By varying the concentration and amounts of the reagents as well as the reaction times, we came to the cycle shown in Table 2. Applying the conditions in Table 2 to octene diol functionalized 1% cross-linked polystyrene, the synthesis of pentamannoside 3 was performed in fourteen hours (Scheme 1). Table 2: The cycle used with trichloroacetimidate donors (25 mol scale) step function reagent Time (min) 1 Kopplen 10 equiv. Donor and 0.5 equiv. TMSOTf 30 2 To wash dichloromethane 6 3 Couple 10 equiv. Donor and 0.5 equiv. TMSOTf 30 4 To wash dichloromethane 6 5 To wash 1: 9 methanol: dichloromethane 6 6 protection 2 × 10 equiv. NaOMe 80 elimination (1: 9 methanol: dichloromethane) 7 To wash 1: 9 methanol: dichloromethane 4 8th To wash 0.2 M acetic acid in tetrahydrofuran 4 9 To wash tetrahydrofuran 6 10 To wash dichloromethane 6

The ability to analyze the resin-bound oligosaccharide is essential for the successful development of solid-phase synthesis. B. Yan, Acc. Chem. Res. 31, 621 (1998). The two-dimensional nuclear magnetic resonance analysis of the resin-bound pentamer 6 was performed by high-resolution Magic Angle-Spinning (HR-MAS-NMR) techniques (see 8th ). Analysis of the NMR spectra revealed characteristic anomeric signals between 97 and 103 ppm. Furthermore, the homonuclear TOCSY HR-MAS analysis confirmed the presence of five unique anomeric protons. For resin TOCSY analyzes of 6 see next example. In accordance with previous experiments, a slight line broadening was observed in the spectra of the resin-bound sample. PH Seeberger, X. Beebe, DG Sukenick, S. Pochapsky, SJ Danishefsky, Angew. Chem. Int. Ed. Engl. 36, 491 (1997). Overall, the HR-MAS NMR data of the polymer-bound pentamer clearly corresponded to an authentic pentamer sample prepared in solution. The remarkable purity of the resin-bound pentasaccharide 6, after 9 synthetic steps without any purification, encouraged us to explore the synthesis of larger structures. Heptamer 4 and Decamer 5 were prepared with average yields of 90-95% per step. The short reaction times, three hours per monomer unit, allowed the synthesis of 4 in 20 hours and an overall yield of 42%. For comparison, we synthesized heptamannoside 4 on the solid support manually in 14 days with an overall yield of 9%.

Example 3

Given the success of the automated - mannoside construction, the fully-protected phytoalexin-inducer (PE) -glucan 7 was chosen as a more complex target (see 9 ). A. Darvill, et al., Glycobiology 2, 181 (1992). The presence of a fungal glucan oligosaccharide in the soybean plant triggers the release of antibiotic phytoalexins. The response in the soybean host plant elicited by the PE glucan is the most widely studied defense mechanism in plants. These oligosaccharides have previously been synthesized in solution and on the solid support and were expected to serve as a benchmark in our automation effort. See P. Fugedi, W. Birberg, PJ Garegg, A. Pilotti, Carb. Res. 164, 297 (1987) and KC Nicolaou, N. Winsisinger, J. Pastor, F. De Roose, J. Am. Chem. Soc. 119, 449 (1997) and references therein.

For the synthesis of the branched, (1 → 3) / (1 → 6) PE structure, we envisioned the use of two different glycosyl phosphate donors, 8 and 9. We recently listed glycosyl phosphates as worthy full glycosylation agents readily made from glycal precursors. OJ Plante, RB Andrade, PH Seeberger, Org Lett 1, 211 (1999). Strategic protecting groups considerations have spurred us to use the levulinoyl ester as a 6-O temporary protecting group and the 2-O-pivaloyl group to ensure complete selectivity in the glycosylation reaction. The deprotection of the levulinoyl ester was achieved with a hydrazine solution in pryridine / acetic acid while the phosphate moiety was activated with TMSOTf.

in the Unlike peptide and nucleic acid synthesis, many will the manipulations involved in oligosaccharide chemistry carried out at room temperature. From the derivation of solution phase studies We were aware that the use of glycosyl phosphates, such as many donors require low temperatures for optimal results would. D. Kahne, S. Walker, X. Cheng, D. Van Enger, J. Am. Chem. Sec. 111, 6811 (1989). To address this requirement, we have one Temperature-controlled reaction vessel designed. The vessel is through a cooling jacket envelops, which is easily connected to a commercial cooling device can be. Demonstrated model reactions with phosphate donor 8 the ease with which a temperature variable in the automation cycle can be installed.

The coupling and deprotection conditions were adjusted for the use of glycosyl phosphates and levulinoyl ester, resulting in the cycle shown in Table 3. Activation of the phosphate donor 8 at -15 ° C required shorter reaction times than required for trichloroacetimidate 2. The levulinate protection elimination occurred at + 15 ° C and required only a fifteen minute reaction time, compared to the longer times often used for acetyl ester cleavage. As in the synthesis of polymannosides 3-5, double glycosylation and double deprotection were used. The incorporation of these modifications into the automated cycle resulted in excellent yield and high purity of a model (1 → 6) trisaccharide. Table 3: The cycle used with phosphate donors (25 mol scale) step function reagent Time (min) 1 coupled 5 equiv. Donor and 5 equiv. TMSOTf 15 2 To wash dichloromethane 6 3 Couple 5 equiv. Donor and 5 equiv. TMSOTf 15 4 watch 1: 9 methanol: dichloromethane 4 5 To wash tetrahydrofuran 4 6 To wash 3: 2 pyridine: acetic acid 3 7 protection elimination 2 x 20 equiv. Hydrazine (3: 2 pyridine: acetic acid 30 8th To wash 3: 2 pyridine: acetic acid 3 9 To wash 1: 9 methanol: dichloromethane 4 10 To wash 0.2 M acetic acid in tetrahydrofuran 4 11 To wash tetrahydrofuran 4 12 To wash dichloromethane 6

• Glycosylierungsbedingungen: 25 μmol Maßstab; 25 μmol Harz (83 mg, 0,30 mmol/g Beladung); 5 Äquiv. Donator 8 oder 9 (90 bzw. 170 mg); 5 Äquiv. TMSOTf (1 ml, 0,125 M TMSOTf in CH₂Cl₂) zweimal für jeweils 15 Minuten bei –15°C wiederholt. Schutzeliminierungsbedingungen: 25 μmol Maßstab; 4 ml, 0,25 M N₂H₄ in Pyridin:Essigsäure (3:2), zweimal für jeweils 15 Minuten bei 15°C wiederholt.

The automated cycle in Table 3 was then applied to the synthesis of more complex PE oligosaccharides using alternating phosphate building blocks (Scheme 2). Branched hexasaccharide 10 was prepared in ten hours with a yield in excess of 80%, as judged by HPLC. We also prepared dodecasaccharide 7 in 17 hours with a yield of over 50% using the same cycle. Remarkably, the solution-phase synthesis of only two phosphate assemblies was required, greatly reducing the manual labor required to assemble a structure of this size. The useful production of material by automation represents a significant improvement over previous methods for polysaccharide synthesis. Scheme 2: Automated oligosaccharide synthesis using glycosyl phosphates

• Glycosylation conditions: 25 μmol scale; 25 μmol resin (83 mg, 0.30 mmol / g loading); 5 equiv. Donor 8 or 9 (90 or 170 mg); 5 equiv. Repeat TMSOTf (1 mL, 0.125 M TMSOTf in CH ₂ Cl ₂ ) twice for 15 minutes each at -15 ° C. Protection elimination conditions: 25 μmol scale; 4 ml, 0.25 MN ₂ H ₄ in pyridine: acetic acid (3: 2), repeated twice for 15 minutes each at 15 ° C.

Example 4

Complex-like trisaccharides

To the development of method for the activation and coupling of anomeric trichloroacetimidate and Glycosyl phosphate donors and for the elimination of acetyl and levulinoyl esters, designed a synthesis that covers all aspects exploits our automated chemistry. We chose trisaccharide 24, which is built on three different monomer units, as a target molecule of and developed a synthesis to different donors and temporary protecting groups incorporate (Scheme 5). Glycans containing this trisaccharide motif are difficult to produce synthetically because of the presence of Gal - - (1 → 4) GlcNAc and GlcNAc - (1 → 2) -manic bonds.

Schema 5: Synthese von Trisaccharid 24 unter Verwendung von Glycosyltrichloracetimidaten und Glycosylphosphaten

Scheme 5: Synthesis of trisaccharide 24 using glycosyl trichloroacetimidates and glycosyl phosphates

Considering the activation and deprotection conditions necessary for the use of donors 11, 22, and 23, we developed a suitable coupling cycle (Table 4). Applying the sequence shown in Table 4, we performed the synthesis of trisaccharide 24 on a 1% cross-linked polystyrene support (Scheme 5). After the 10 hour coupling cycle, the trisaccharide 24 was cleaved from the support and analyzed by HPLC to give 24 with an overall yield of 60%. We were encouraged by this result, as it was the first example that included all of the automated protocols in a single coupling cycle. Further, the successful deprotection of a levulinate ester in the presence of a phthaloylamine protecting group provided an orthogonal protecting group strategy which generally can be applied to other sequences. It is understandable that any degree of orthogonality developed for our automated protocol will increase the speed and efficiency with which increasingly complex carbohydrates can be produced. Table 4. The cycle used for the synthesis of trisaccharide 24. step function reagent Time (min) 1 Couple 4 equiv. Donor 11 and 0.4 equiv. TMSOTf 30 2 To wash dichloromethane 6 3 Couple 4 equiv. Donor 11 and 0.4 equiv. TMSOTf 30 4 To wash dichloromethane 6 5 To wash 1: 9 methanol: dichloromethane 4 6 protection elimination 2 × 10 equiv. NaOMe (1: 9 methanol: dichloromethane) 80 7 To wash 1: 9 methanol: dichloromethane 4 8th To wash 0.2 M acetic acid in tetrahydrofuran 4 9 To wash tetrahydrofuran 4 10 To wash dichloromethane 6 11 Couple 4 equiv. Donor 22 and 0.4 equiv. TMSOTf 30 12 To wash dichloromethane 6 13 Couple 4 equiv. Donor 22 and 0.4 equiv. TMSOTf 30 14 To wash dichloromethane 6 15 To wash 1: 9 methanol: dichloromethane 4 16 To wash tetrahydrofuran 4 17 To wash 3: 2 pyridine: acetic acid 3 18 protection elimination 2 x 20 equiv. Hydrazine (3: 2 pyridine: acetic acid) 30 19 To wash 3: 2 pyridine: acetic acid 3 20 To wash 1: 9 methanol: dichloromethane 4 21 To wash 0.2 M acetic acid in tetrahydrofuran 4 22 To wash tetrahydrofuran 4 23 To wash dichloromethane 6 24 Couple 5 equiv. Donator 23 and 5 equiv. TMSOTf 15 25 26 Washing coupling Dichloromethane 5 equiv. Donator 23 and 5 equiv. TMSOTf 6 15 27 To wash 1: 9 methanol: dichloromethane 4 28 To wash tetrahydrofuran 4 29 To wash dichloromethane 6

Phytoalex inducer-glucans Hexasaccharide 10 and dodecasaccharide 7 were in a rapid way and Manner using glycosyl phosphates. We expect, that this technology is the fast and reliable procurement of synthetic Oligosaccharides by a non-specialist without undue difficulty enable becomes. The release from the tedious manual work in Carbohydrate synthesis will be the gateway to synthetic Material for promote biochemical investigations. The ease of getting defined structures from one Device becomes affect the field of glycobiology, so we one day are able to understand the importance of oligosaccharides and glycoconjugates fully appreciate in nature.

Example 5

Synthesis and characterization of building units 8 and 9

Scheme I. Synthesis of building blocks 8 and 9

SYNTHESIS OF DIBUTYL 3,4-DI-O-BENZYL-6-O-LEVULINOYL-2-O-PIVALOYL-D-GLUCOPYRANOSYL PHOSPHATE B.

3,4-Di-O-benzyl-6-O-levulinoyl-D-arabino-hex-1-enol (2.12 g, 5.0 mmol) was dissolved in CH ₂ Cl ₂ (5 mL) and cooled to 0 °. A 0.08 M dimethyldioxirane solution in acetone (75 mL, 6.0 mmol) was added and the reaction mixture was stirred for 15 minutes. After the solvent was removed in a stream of N ₂ and the remaining residue was dried in vacuo for 15 minutes at 0 ° C, 20 ml of CH ₂ Cl _{2 were} added. The solution was cooled to 78 ° C for 15 minutes and dibutyl phosphate (1.04 mL, 5.25 mmol) was then added dropwise over 5 minutes. After complete addition, the reaction mixture was mentioned at 0 ° C and DMAP (2.44 g, 20.0 mmol) and pivaloyl chloride (1.23 mL, 10.0 mmol) were added. The solution was warmed to room temperature over an hour. The solvent was removed in vacuo and the residue was purified by flash column chromatography on silica gel to afford 3.29 g (90%) of 8 as a colorless oil. ¹ H NMR (500 MHz) 7.34-7.24 (m, 10H), 5.23 (app t, J = 7.6, 7.9 Hz, 1H), 5.14 (app t, J = 7.9, 9.2 Hz, 1H), 4.81-4.78 (m, 2H), 4.72 (d, J = 11.0 Hz, 1H), 4.57 (d, J = 10 , 7Hz, 1H), 4.39 (d, J = 11.3Hz, 1H), 4.23 (dd, J = 4.0, 12.2Hz, 1H), 3.67-3.66 (m, 2H), 2.76-2.70 (m, 2H), 2.57-2.54 (m, 2H), 2.19 (s, 3H), 1.67-1.60 (m , 4H), 1.41-1.35 (m, 4H), 1.20 (s, 9H), 0.96-0.90 (m, 6H); ¹³ C NMR (125 MHz) 206.4, 177.0, 172.5, 137.9, 137.5, 128.7, 128.6, 128.4, 128.3, 128.2, 128.0 , 127.5, 96.6, 96.6, 82.9, 75.3, 75.2, 73.8, 72.9, 68.2, 68.2, 68.0, 68.0, 62 , 7, 39.0, 38.0, 32.3, 32.3, 32.3, 32.2, 30.0, 27.9, 27.3, 18.8, 18.8, 13.8 , 13.7; ³¹ PNMR (120 MHz) -1.66.

Synthesis of dibutyl (2,3,4,6-tetra-O-benzyl) -D-glucopyranosyl) - (1 → 3) -4-O-benzyl-4-6-O-levulinoyl-2-O-pivaloyl- -D-glucopyranosyl phosphate 9.

2,3,4,6-tetra-O-benzyl-D-glucopyranosyl- (1 → 3) -4-O-benzyl-6-O-levulinoyl-D-arabino-hex-1-enitol (1.00 g, 1.14 mmol) was dissolved in CH ₂ Cl ₂ (5 mL) and cooled to 0 ° C. A 0.08 M dimethyldioxirane solution in acetone (25 mL, 1.77 mmol) was added and the reaction mixture was stirred for 15 minutes. After the solvent was removed in a stream of N ₂ and the remaining residue was dried in vacuo for 15 minutes at 0 ° C, 20 ml of CH ₂ Cl _{2 were} added. The solution was cooled to -78 ° C for 15 minutes and dibutyl phosphate (0.26 mL, 1.30 mmol) was then added dropwise over 5 minutes. After complete addition, the reaction mixture was warmed to 0 ° C and DMAP (0.57 g, 4.72 mmol) and pivaloyl chloride (0.29 mL, 2.36 mmol) were added. The solution was left on for 1 hour Room temperature warmed up. The solvent was removed in vacuo and the residue was purified by flash column chromatography on silica gel to give 950 mg (70%) of 9 as a colorless oil. ¹ H NMR (500 MHz) 7.32-7.24 (m, 25H), 5.22 (app t, J = 7.9, 9.4 Hz, 1H), 5.14 (app t, J = 6.4, 7.9 Hz, 1H), 5.01 (d, J = 11.0 Hz, 1H), 4.93 (d, J = 11.6 Hz, 1H), 4.87 (d, J = 11.0 Hz, 1H), 4.81 (d, J = 10.7 Hz, 1H), 4.74 (d, J = 11.0 Hz, 1H), 4.67 (d, J = 7.9Hz, 1H), 4.64-4.54 (m, 3H), 4.48 (s, 2H), 4.38-4.36 (m, 1H), 4.23 (dd, J = 4.3, 11.9 Hz, 1H), 4.18-4.11 (m, 2H), 4.07-3.98 (m, 4H), 3.80 (d, J = 11.0 Hz, 1H), 3.64-3.54 (m, 4H), 2.20 (s, 3H), 1.63-1.61 (m, 4H), 1.40-1.35 (m, 4H), 1.23 (s, 9H), 0.95-0.90 (m, 6H); ¹³ C NMR (125 MHz) 206.5, 176.6, 172.5, 171.4, 138.6, 138.5, 138.1, 129.0, 128.9, 128.8, 128.6 , 128.5, 128.5, 128.3, 128.3, 128.1, 128.0, 127.7, 127.7, 127.6, 103.5, 96.5, 96.5, 84 , 8, 82.6, 9.2, 78.2, 75.9, 75.4, 75.2, 75.1, 75.0, 73.8, 73.6, 73.6, 73.2 , 73.1, 69.3, 68.3, 68.2, 68.0, 67.9, 62.9, 60.6, 39.0, 38.0, 32.3, 32.3, 32 , 3, 30.0, 27.9, 27.5, 27.4, 27.3, 21.3, 18.8, 18.8, 14.4, 13.8, 13.8; ³¹ P NMR (120 MHz) -2.33.

Example 6

Evaluation of the optimal temperature for phosphate glycosylation

Scheme II. Trisaccharide synthesis at different temperatures

Trisaccharide synthesis was carried out at room temperature and at -15 ° C using the following conditions. 25 μmol Scale: 25 μmol resin (83 mg, 0.30 mmol / g loading); 5 equiv. Dunator 8 (90 mg); 5 equiv. TMSOTf (1 ml, 0.125 M TMSOTf in CH ₂ Cl ₂ ) for 15 minutes. Protection elimination conditions: 25 μmol Scale: 4 ml, 0.25 MN ₂ H ₄ in pyridine: acetic acid (3: 2). HPLC analysis of the fission products of room temperature synthesis and -15 ° C synthesis are in 5 respectively. 6 shown.

• Analytisches HPLC Chromatogramm der Raumtemperatur Triglucosid-Synthese, anschließend an die Harzspaltung. Fließrate = 1 ml/min, 20→30% EtOAc/Hexane (20 Minuten): 5 Minuten = Trimer.

HPLC data for trimer synthesis using phosphate 8 at room temperature (Scheme II):

• Analytical HPLC chromatogram of the room temperature triglucoside synthesis, subsequent to the resin cleavage. Flow rate = 1 ml / min, 20 → 30% EtOAc / hexanes (20 minutes): 5 minutes = trimer.

• Analytisches HPLC Chromatogramm der –15°C Triglucosid-Synthese, anschließend an die Harzspaltung. Fließrate = 1 ml/min, 20→30% EtOAc/Hexane (20 Minuten); 5 Minuten = Trimer.

HPLC data for trimer synthesis using phosphate 8 at -15 ° C (Scheme II):

• Analytical HPLC Chromatogram of the -15 ° C triglucoside synthesis, subsequent to the resin cleavage. Flow rate = 1 ml / min, 20 → 30% EtOAc / hexanes (20 minutes); 5 minutes = trimer.

equivalent

professionals will recognize, or be made use of, nothing more than routine experimentation in the Be able to many equivalents to the specific embodiments to determine the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims (39)

Device for the automated solid-phase synthesis of oligosaccharides, which Includes: a reaction vessel with at least one insoluble Resin beads; at least a donor vessel with a Sacchariddonatorlösung; at least an activator vessel with a Assets Torre agent solution; at least a deblocking vessel with a deblocking; at least a solvent vessel with a Solvents; one Solution transfer system which is capable of the saccharide donor solution, activator reagent solution, deblocking reagent solution and the solvent transferred to; and a computer for controlling the solution transfer system. Apparatus according to claim 1, wherein the at least an insoluble one resin beads having a glycosyl acceptor via an organic linker is bound to this. Apparatus according to claim 1 or 2, which further a temperature control unit for controlling the temperature of Reaction vessel has. Apparatus according to claim 3, wherein the temperature control unit controlled by the computer. Apparatus according to claim 3 or 4, wherein the temperature control unit measures the internal temperature of the reaction vessel. A device according to claim 3, 4 or 5, wherein the Reaction vessel a double-walled Structure is which two cavities forms, wherein in the first cavity, the synthesis of the oligosaccharides takes place, and wherein the second cavity is a coolant of the temperature control unit contains. Apparatus according to claim 6, wherein the double-walled Structure of the reaction vessel from Glass exists. Device according to one of claims 3 to 7, wherein the temperature control unit is able to keep the reaction vessel at a temperature between -80 ° C and + 60 ° C. Apparatus according to claim 8, wherein the temperature control unit is able to keep the reaction vessel at a temperature between -25 ° C and + 40 ° C. Device according to one of the preceding claims, wherein the at least one donor device contains a solution which a glycosyl trichloroacetimidate or a glycosyl phosphate. Device according to one of the preceding claims, wherein the at least one activator vessel contains a solution which a Lewis acid contains. Apparatus according to claim 11, wherein the at least an activator vessel contains a solution containing a Silyltrifluormethansulfonat contains. Apparatus according to claim 12, wherein said at least an activator vessel containing a solution containing trimethylsilyl trifluoromethanesulfonate contains. Device according to one of the preceding claims, wherein the at least one deblocking vessel contains a solution containing sodium methoxide or hydrazine. Device according to one of the preceding claims, wherein the at least one solvent vessel dichloromethane, Contains tetrahydrofuran and / or methanol. The apparatus of claim 15, wherein a first solvent vessel is dichloromethane contains a second solvent vessel methanol contains and a third solvent vessel tetrahydrofuran contains. Device according to one of the preceding claims, which further comprises at least one blocking vessel containing a blocking reagent solution. Apparatus according to claim 17, wherein said at least a blocking vessel contains a solution which Benzyl trichloroacetimidate or a carboxylic acid. The device of claim 18, wherein the carboxylic acid is levulinic acid. The apparatus of claim 19, wherein a first solvent vessel is dichloromethane contains a second solvent vessel methanol contains and a third solvent vessel tetrahydrofuran contains; a fourth solvent vessel a solution contains which pyridine and acetic acid and a fifth Solvent vessel a 0.2 M solution acetic acid in tetrahydrofuran. The device of claim 19, wherein a first donor vessel contains a solution which Glycosyltrichloracetimidat, a second donor vessel containing a solution which a first glycosyl phosphate, a third donor vessel containing a solution which a second glycosyl phosphate containing at least one activator vessel a solution which Trimethylsilyltrifluormethansulfonat, a first Deblockierungsgefäß contains a solution which Hydrazine, a second deblocking vessel containing a solution containing sodium methoxide comprising a first solvent vessel of dichloromethane contains a second solvent vessel methanol contains and a third solvent vessel tetrahydrofuran contains a fourth solvent vessel contains a solution which Pyridine and acetic acid and a fifth Solvent vessel a 0.2 M solution acetic acid in tetrahydrofuran. Device according to one of the preceding claims, wherein the at least one insoluble resin beads consists of an octenediol-functionalized resin. Device according to one of claims 2 to 22, wherein the organic Left consists of a glycosyl phosphate. Process for forming a carbon-heteroatom bond between a glycosyl donor and a substrate which is in solution in the Reaction vessel of a Device according to one of the preceding claims, the step of combining a glycosyl donor having a reactive anomeric carbon, a substrate having a heteroatom which has a Carries hydrogen, and an activating reagent, wherein the activating reagent activates the reactive anomeric carbon of the glycosyl donor, thereby forming a product having a carbon-heteroatom bond between the anomeric carbon of the glycosyl donor and the heteroatom of the substrate. The method of claim 24, wherein the glycosyl donor, which has a reactive anomeric carbon, from the group consisting of glycosyl phosphates, glycosyl phosphites, glycosyl trichloroacetimidates, Glycosyl halides, glycosyl sulfides, glycosyl sulfoxides, n-pentenyl glycosides and 1,2-anhydroglycosides is. A method according to claim 24 or 25, wherein the heteroatom, which carries a hydrogen of the substrate, consisting of the group is selected from oxygen, nitrogen and sulfur. The method of claim 24, 25 or 26, wherein said Activation reagent is a Lewis acid is. A method according to any one of claims 24 to 27, wherein the activating reagent a silyl trifluoromethanesulfonate. The method of claim 28, wherein the activating reagent Trimethylsilyltrifluormethansulfonat is. Method according to one of claims 24 to 29, wherein the substrate, which has a heteroatom which carries a hydrogen, via a Covalent linker is attached to a solid support. A method according to any one of claims 24 to 30, wherein the glycosyl donor, which has a reactive anomeric carbon via a Covalent linker is attached to a solid support. The method of claim 30 or 31, wherein the covalent linker is -O- (CH ₂ ) ₃ CH = CH (CH ₂ ) ₃ -O-. The method of claim 32, wherein the solid support comprises resin beads is. The method of claim 33, wherein the substrate, which has a heteroatom which carries a hydrogen from the group consisting of monosaccharides, oligosaccharides, polysaccharides and glycoconjugates selected is. The method of any one of claims 24 to 34, which further the steps of applying positive pressure or a vacuum the reaction vessel of the device includes, whereby the liquid Phase from the reaction vessel of the device is removed, as well as the addition of solvent to the reaction vessel of the device. The method of claim 35, further comprising the step the treatment of the product in the reaction vessel of the device with a solution comprising a reagent for removing the protection, thereby a protective group is removed from the product to a second To produce a product which has a heteroatom, which is a Carries hydrogen, the second product over a covalent linker is attached to a solid support. The method of claim 36, which is further in solution in the reaction vessel of the device the step of combining a glycosyl donor, which one having reactive anomeric carbon, wherein the second product has a heteroatom which carries a hydrogen, with an activating reagent, wherein the activating reagent is the reactive one anomeric carbon of the glycosyl donor activated, creating a third product is formed, which is a carbon-heteroatom bond between the anomeric carbon of the glycosyl donor and the heteroatom of the second product, wherein the third product via a Covalent linker is attached to a solid support. The method of claim 35, further comprising the step the treatment of the product in the reaction vessel of the device with a solution comprising a conversion reagent for producing a second Having a product which is a reactive anomeric carbon wherein the second product is via a covalent linker to a solid support is bound. The method of claim 38, which is further in solution in the reaction vessel of the device the step of combining a substrate which is a heteroatom which carries a hydrogen, wherein the second product having a reactive anomeric carbon with an activating reagent wherein the activating reagent is the reactive anomeric carbon of the second product, thereby forming a third product which is a carbon-heteroatom bond between the anomeric Carbon of the second product and the heteroatom of the substrate wherein the third product is via a covalent linker to a solid support is bound.